Diagnosis and treatment of ALP related disorders

ABSTRACT

The present invention relates to Alp polypeptides, nucleic acids encoding such polypeptides, cells, tissues and animals containing such nucleic acids, antibodies to such polypeptides, assays utilizing such polypeptides, and methods relating to all of the foregoing. Methods for treatment, diagnosis, and screening are provided for Alp related diseases or conditions characterized by an abnormal interaction between an Alp polypeptide and an Alp binding partner.

RELATED APPLICATIONS

[0001] This application claims priority to the U.S. Provisional PatentApplication No. 60/049,477, by Plowman et al., entitled “Diagnosis andTreatment of ALP Related Disorders,” and filed Jun. 12, 1997, which isincorporated herein by reference in its entirety, including anydrawings.

FIELD OF THE INVENTION

[0002] The present invention relates to tyrosine phosphatases. Inparticular, the invention concerns a protein we have named ALP (AdaptorLike Phosphatase), nucleotide sequences encoding ALP, various productsand assay methods that can be used for identifying compounds useful forthe diagnosis and treatment of various ALP-related diseases andconditions, for example cell proliferative disorders.

BACKGROUND OF THE INVENTION

[0003] The following description is provided to aid in understanding theinvention but is not admitted to be prior art to the invention.

[0004] Cellular signal transduction is a fundamental mechanism wherebyexternal stimuli that regulate diverse cellular processes are relayed tothe interior of cells. One of the key biochemical mechanisms of signaltransduction involves the reversible phosphorylation of proteins, whichenables regulation of the activity of mature proteins by altering theirstructure and function. The best characterized protein kinases ineukaryotes phosphorylate proteins on the alcohol moiety of serine,threonine and tyrosine residues. These kinases largely fall into twogroups, those specific for phosphorylating serines and threonines, andthose specific for phosphorylating tyrosines.

[0005] The phosphorylation state of a given substrate is also regulatedby a class of proteins responsible for removal of the phosphate groupadded to a given substrate by a protein kinase. The protein phosphatasescan also be classified as being specific for either serine/threonine ortyrosine. The known enzymes can be divided into two groups—receptor andnon-receptor type proteins. Most receptor-type protein tyrosinephosphatases (RPTPs) contain two conserved catalytic tyrosinephosphatase domains each of which encompasses a segment of 240 aminoacid residues (Saito et al; Cell Growth and Diff. 2:59-65, 1991). TheRPTPs can be subclassified further based upon the amino acid sequencediversity of their extracellular domains (Saito, et al; supra; Krueger,et al; Proc. Natl. Acad. Sci. USA 89:7417-7421, 1992).

[0006] Alignment of primary peptide sequences of both types of knownPTPases shows some sequence consensus in catalytic domains and has madeit possible to identify cDNAs encoding proteins with tyrosine phosphateactivity via the polymerase chain reaction (PCR).

[0007] Many kinases and phosphatases are involved in regulatory cascadeswherein their substrates may include, but are not limited to, otherkinases and phosphatases whose activities are regulated by theirphosphorylation state. Ultimately the activity of some downstreameffector is modulated by phosphorylation resulting from activation ofsuch a pathway.

[0008] Tyrosine phosphatases have been thought to be possible candidatecancer causing proteins. Inappropriate activity through overexpressionof RPTP-alpha, for example, has been associated with colon cancer(Pallen, et al, WO 94/01119, published Jan. 20, 1994). A need exists toidentify additional proteins whose inappropriate activity may lead tocancer or other disorders so that pharmaceutical compounds for thetreatment of those disorders might also be identified.

SUMMARY OF THE INVENTION

[0009] Disclosed herein is a tyrosine phosphatase overexpressed incancer cells which we have named ALP. The properties of ALP aredescribed below. The present invention concerns ALP polypeptides,nucleic acids encoding such polypeptides, cells, tissues and animalscontaining such nucleic acids, antibodies to the polypeptides, assaysutilizing the polypeptides, and methods relating to all of theforegoing.

[0010] A first aspect of the invention features an isolated, enriched,or purified nucleic acid molecule encoding an ALP polypeptide.

[0011] By “isolated” in reference to nucleic acid it is meant a polymerof 14, 17, 21 or more nucleotides conjugated to each other, includingDNA or RNA that is isolated from a natural source or that issynthesized. The isolated nucleic acid of the present invention isunique in the sense that it is not found in a pure or separated state innature. Use of the term “isolated” indicates that a naturally occurringsequence has been removed from its normal cellular (i.e., chromosomal)environment. Thus, the sequence may be in a cell-free solution or placedin a different cellular environment. The term does not imply that thesequence is the only nucleotide sequence present, but that it isessentially free (about 90-95% pure at least) of nucleotide materialnaturally associated with it and thus is meant to be distinguished fromisolated chromosomes.

[0012] By the use of the term “enriched” in reference to nucleic acid itis meant that the specific DNA or RNA sequence constitutes asignificantly higher fraction (2-5 fold) of the total DNA or RNA presentin the cells or solution of interest than in normal or diseased cells orin the cells from which the sequence was taken. This could be caused bya person by preferential reduction in the amount of other DNA or RNApresent, or by a preferential increase in the amount of the specific DNAor RNA sequence, or by a combination of the two. However, it should benoted that “enriched” does not imply that there are no other DNA or RNAsequences present, just that the relative amount of the sequence ofinterest has been significantly increased.

[0013] The term “significant” here is used to indicate that the level ofincrease is useful to the person making such an increase, and generallymeans an increase relative to other nucleic acids of about at least 2fold, more preferably at least 5 to 10 fold or even more. The term alsodoes not imply that there is no DNA or RNA from other sources. The othersource DNA may, for example, comprise DNA from a yeast or bacterialgenome, or a cloning vector such as pUC19. This term distinguishes thesequence from naturally occurring enrichment events, such as viralinfection, or tumor type growths, in which the level of one mRNA may benaturally increased relative to other species of mRNA. That is, the termis meant to cover only those situations in which a person has intervenedto elevate the proportion of the desired nucleic acid.

[0014] It is also advantageous for some purposes that a nucleotidesequence be in purified form. The term “purified” in reference tonucleic acid does not require absolute purity (such as a homogeneouspreparation); instead, it represents an indication that the sequence isrelatively purer than in the natural environment (compared to thenatural level this level should be at least 2-5 fold greater, e.g., interms of mg/mL). Individual clones isolated from a cDNA library may bepurified to electrophoretic homogeneity. The claimed DNA moleculesobtained from these clones can be obtained directly from total DNA orfrom total RNA. The cDNA clones are not naturally occurring, but ratherare preferably obtained via manipulation of a partially purifiednaturally occurring substance (messenger RNA). The construction of acDNA library from mRNA involves the creation of a synthetic substance(cDNA) and pure individual cDNA clones can be isolated from thesynthetic library by clonal selection of the cells carrying the cDNAlibrary. Thus, the process which includes the construction of a cDNAlibrary from mRNA and isolation of distinct cDNA clones yields anapproximately 10-fold purification of the native message. Thus,purification of at least one order of magnitude, preferably two or threeorders, and more preferably four or five orders of magnitude isexpressly contemplated. The term is also chosen to distinguish clonesalready in existence which may encode ALP but which have not beenisolated from other clones in a library of clones. Thus, the term coversclones encoding ALP which are isolated from other non-ALP clones.

[0015] The term “nucleic acid molecule” describes a polymer ofdeoxyribonucleotides (DNA) or ribonucleotides (RNA). The nucleic acidmolecule may be isolated from a natural source by cDNA cloning orsubtractive hybridization or synthesized manually. The nucleic acidmolecule may be synthesized manually by the triester synthetic method orby using an automated DNA synthesizer.

[0016] The term “cDNA cloning” refers to hybridizing a small nucleicacid molecule, a probe, to genomic cDNA. The probe hybridizes (binds) tocomplementary sequences of cDNA.

[0017] The term “complementary” describes two nucleotides that can formmultiple favorable interactions with one another. For example, adenineis complementary to thymine as they can form two hydrogen bonds.Similarly, guanine and cytosine are complementary since they can formthree hydrogen bonds. Thus if a nucleic acid sequence contains thefollowing sequence of bases, thymine, adenine, guanine and cytosine, a“complement” of this nucleic acid molecule would be a moleculecontaining adenine in the place of thymine, thymine in the place ofadenine, cytosine in the place of guanine, and guanine in the place ofcytosine. Because the complement can contain a nucleic acid sequencethat forms optimal interactions with the parent nucleic acid molecule,such a complement can bind with high affinity to its parent molecule.

[0018] The term “hybridize” refers to a method of interacting a nucleicacid sequence with a DNA or RNA molecule in solution or on a solidsupport, such as cellulose or nitrocellulose. If a nucleic acid sequencebinds to the DNA or RNA molecule with high affinity, it is said to“hybridize” to the DNA or RNA molecule. The strength of the interactionbetween the probing sequence and its target can be assessed by varyingthe stringency of the hybridization conditions. Under highly stringenthybrydization conditions only highly complementary nucleic acidsequences hybridize. Preferably, such conditions prevent hybridizationof nucleic acids having one or two mismatches out of 20 contiguousnucleotides.

[0019] Various low or high stringency hybridization conditions may beused depending upon the specificity and selectivity desired. Stringencyis controlled by varying salt or denaturant concentrations. Examples ofhybridization conditions are shown in the examples below. High stringentconditions may mean conditions that are at least as stringent as thefollowing: hybridization in 50% formamide, 5× SSC, 50 mM NaH₃PO₄, pH6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5× Denhartsolution at 42° C. overnight; washing with 2× SSC, 0.1% SDS at 45° C.;and washing with 0.2× SSC, 0.1% SDS at 45° C. Those skilled in the artwill recognize how such conditions can be varied to vary specificity andselectivity.

[0020] An ALP polypeptide can be encoded by a full-length nucleic acidsequence or any portion of the full-length nucleic acid sequence. Inpreferred embodiments the isolated nucleic acid comprises, consistsessentially of, or consists of a nucleic acid sequence set forth in SEQID NO:1, a nucleic acid sequence that hybidizes to the nucleic acidsequence set forth in SEQ ID NO:1 or a functional derivative (as definedbelow) of either. The nucleic acid may be isolated from a natural sourceby cDNA cloning or subtractive hybridization; the natural source may bemammalian (human) blood, semen, or tissue and the nucleic acid may besynthesized by the triester or other method or by using an automated DNAsynthesizer.

[0021] The term “mammalian” refers to such organisms as mice, rats,rabbits, goats, more preferably monkeys and apes, and most preferablyhumans.

[0022] In other preferred embodiments, the nucleic acid molecule of theinvention comprises a nucleotide sequence that (a) encodes a polypeptidehaving the full length amino acid sequence set forth in SEQ ID NO:2; (b)is the complement of the nucleotide sequence of (a);

[0023] (c) hybridizes under highly stringent conditions to thenucleotide molecule of (a) and encodes a naturally occurring ALPpolypeptide; (d) encodes an ALP polypeptide having the full length aminoacid sequence of the sequence set forth in SEQ ID NO:2, except that itlacks one or more of the following segments of amino acid residues:1-857, 353-777, 858-1096, 1097-1274, 1101-1214 of SEQ ID NO:2; (e) isthe complement of the nucleotide sequence of (d); (f) encodes apolypeptide having the amino acid sequence set forth in SEQ ID NO:2 fromamino acid residues 1-857, 353-777, 858-1096, 1097-1274, 1101-1214 ofSEQ ID NO:2; (g) is the complement of the nucleotide sequence of (f);(h) encodes a polypeptide having the full length amino acid sequence setforth in SEQ ID NO:2, except that it lacks one or more of the domainsselected from the group consisting of an N-terminal domain, anN-terminal proline-rich domain, a catalytic domain, a C-terminalproline/serine-rich domain, and a C-terminal domain; or (i) is thecomplement of the nucleotide sequence of (h). The nucleic acid moleculeof the invention is isolated, enriched, or purified from, preferably, amammal, or most preferably from a human.

[0024] In yet other preferred embodiments the nucleic acid is anisolated conserved or unique region, for example those useful for thedesign of hybridization probes to facilitate identification and cloningof additional polypeptides, or for the design of PCR probes tofacilitate cloning of additional polypeptides.

[0025] By “conserved nucleic acid regions”, it is meant regions presenton two or more nucleic acids encoding an ALP polypeptide, to which aparticular nucleic acid sequence can hybridize under lower stringencyconditions. Examples of lower stringency conditions suitable forscreening for nucleic acids encoding ALP polypeptides are provided inAbe, et al. J. Biol. Chem. 19:13361 (1992) (hereby incorporated byreference herein in its entirety, including any drawings). Preferably,conserved regions differ by no more than 5 out of 20 continguousnucleotides.

[0026] By “unique nucleic acid region” it is meant a sequence present ina full length nucleic acid coding for an ALP polypeptide that is notpresent in a sequence coding for any other known naturally occurringpolypeptide. Such regions preferably comprise 14, 17, 21 or morecontiguous nucleotides present in the full length nucleic acid encodingan ALP polypeptide. In particular, a unique nucleic acid region ispreferably of human origin.

[0027] In yet another aspect, the invention relates to a nucleic acidvector comprising a nucleic acid molecule encoding an ALP polypeptideand a promoter element effective to initiate transcription in a hostcell.

[0028] The term “nucleic acid vector” relates to a single or doublestranded circular nucleic acid molecule that can be transfected ortransformed into cells and replicate independently or within the hostcell genome. A circular double stranded nucleic acid molecule can be cutand thereby linearized upon treatment with restriction enzymes. Anassortment of vectors, restriction enzymes, and the knowledge of thenucleotide sequences that the restriction enzymes operate upon arereadily available to those skilled in the art. A nucleic acid moleculeof the invention can be inserted into a vector by cutting the vectorwith restriction enzymes and ligating the two pieces together.

[0029] Many techniques are available to those skilled in the art tofacilitate transformation or transfection of the expression constructinto a prokaryotic or eukaryotic organism. The terms “transformation”and “transfection” refer to methods of inserting an expression constructinto a cellular organism. These methods involve a variety of techniques,such as treating the cells with high concentrations of salt, an electricfield, or detergent, to render the host cell outer membrane or wallpermeable to nucleic acid molecules of interest.

[0030] The term “promoter element” describes a nucleotide sequence thatis incorporated into a vector that, once inside an appropriate cell, canfacilitate transcription factor and/or polymerase binding and subsequenttranscription of portions of the vector DNA into mRNA. The. promoterelement precedes the 5′ end of the ALP nucleic acid molecule such thatthe latter is transcribed into mRNA. Host cell machinery then translatesmRNA into a polypeptide.

[0031] Those skilled in the art would recognize that a nucleic acidvector can contain many other nucleic acid elements besides the promoterelement and the ALP nucleic acid molecule. These other nucleic acidelements include, but are not limited to, origins of replication,ribosomal binding sites, nucleic acid sequences encoding drug resistanceenzymes or amino acid metabolic enzymes, and nucleic acid sequencesencoding secretion signals, periplasm or peroxisome localizationsignals, or signals useful for polypeptide purification.

[0032] The invention also features a nucleic acid probe for thedetection of a nucleic acid encoding an Alp polypeptide in a sample. Theterm “nucleic acid probe” refers to a nucleic molecule that iscomplementary to and can bind a nucleic acid sequence encoding the aminoacid sequence substantially similar to that set forth in SEQ ID NO:2.

[0033] The nucleic acid probe contains nucleic acid that will hybridizespecifically to a sequence of at least 14, preferably 17, 20 or 22,continguous nucleotides set forth in SEQ ID NO:1 or a functionalderivative thereof. The probe is preferably at least 14, 17 or morebases in length and selected to hybridize specifically to a uniqueregion of an ALP endocing nucleic acid.

[0034] In preferred embodiments the nucleic acid probe hybridizes tonucleic acid encoding at least 14 contiguous amino acids of thefull-length sequence set forth in SEQ ID NO:1 or a functional derivativethereof. Various low or high stringency hybridization conditions may beused depending upon the specificity and selectivity desired. Underhighly stringent hybridization conditions only highly complementarynucleic acid sequences hybridize. Preferably, such conditions preventhybridization of nucleic acids having 1 or 2 mismatches out of 20contiguous nucleotides.

[0035] Methods for using the probes include detecting the presence oramount of ALP RNA in a sample by contacting the sample with a nucleicacid probe under conditions such that hybridization occurs and detectingthe presence or amount of the probe bound to ALP RNA. The nucleic acidduplex formed between the prcbe and a nucleic acid sequence coding foran ALP polypeptide may be used in the identification of the sequence ofthe nucleic acid detected (for example see, Nelson et al., inNonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego(Kricka, ed., 1992) hereby incorporated by reference herein in itsentirety, including any drawings). Kits for performing such methods maybe constructed to include a container having disposed therein a nucleicacid probe.

[0036] Another feature of the invention is a nucleic acid molecule asset forth in SEQ ID NO:1 or fragments thereof, comprising one or moreregions that encode an ALP polypeptide or an ALP domain polypeptide,where the ALP polypeptide or the ALP domain polypeptide is fused to anon-ALP polypeptide. Such fused polypeptides include, for example, butare not limited to, a GST-fusion protein.

[0037] The invention also features recombinant nucleic acid, preferablyin a cell or an organism. The recombinant nucleic acid may contain asequence set forth in SEQ ID NO:1 or a functional derivative thereof anda vector or a promoter effective to initiate transcription in a hostcell. The recombinant nucleic acid can alternatively contain atranscriptional initiation region functional in a cell, a sequencecomplimentary to an RNA sequence encoding an ALP polypeptide and atranscriptional termination region functional in a cell.

[0038] Another aspect of the invention relates to a recombinant cell ortissue comprising a nucleic acid molecule encoding an ALP polypeptide.The recombinant cell may comprise a nucleic acid molecule encodingeither an ALP polypeptide; an ALP domain polypeptide; or an ALPpolypeptide or ALP domain polypeptide fused to a non-ALP polypeptide.

[0039] The term “recombinant organism” refers to an organism that has anew combination of genes or nucleic acid molecules. A new combination ofgenes or nucleic acid molecules can be introduced to an organism using awide array of nucleic acid manipulation techniques available to thoseskilled in the art.

[0040] The term “organism” relates to any living being comprised of aleast one cell. An organism can be as simple as one eukaryotic cell oras complex as a mammal. Therefore, a recombinant organism can also be arecombinant cell, which may be a eukaryotic or a prokaryotic organism.

[0041] The term “eukaryote” refers to an organism comprised of cellsthat contain a nucleus. Eukaryotes are differentiated from “prokaryotes”which do not have a nucleus and lack other cellular structures found ineukaryotes, such as mitochondria and endoplasmic reticulum. Prokaryotesinclude unicellular organisms, such as bacteria, while eukaryotes arerepresented by yeast, invertebrates, and vertebrates.

[0042] The recombinant cell can harbor a nucleic acid vector that isextragenomic. The term “extragenomic” refers to a nucleic acid vectorwhich does not insert into the cell genome. Many nucleic acid vectorsare designed with their own origins of replication allowing them toutilize the recombinant cell replication machinery to copy and propagatethe vector nucleic acid sequence. These vectors are small enough thatthey are not likely to harbor nucleic acid sequences homologous togenomic sequences of the recombinant cell. Thus these vectors replicateindependently of the host genome and do not recombine with or integrateinto the genome.

[0043] A recombinant cell can harbor a portion of a nucleic acid vectorin an intragenomic fashion. The term “intragenomic” defines a nucleicacid construct that is incorporated within the cell genome. Multiplenucleic acid vectors available to those skilled in the art containnucleic acid sequences that are homologous to nucleic acid sequences ina particular organism's genomic DNA. These homologous sequences willresult in recombination events that integrate portions of the vectorinto the genomic DNA. Those skilled in the art can control which nucleicacid sequences of the vector are integrated into the cell genome byflanking the portion to be incorporated into the genome with homologoussequences in the vector.

[0044] Another aspect of the invention features an isolated, enriched,or purified ALP polypeptide.

[0045] By “ALP polypeptide” it is meant an amino acid sequencesubstantially similar to the sequence shown in SEQ ID NO:2, or fragmentsthereof. A sequence that is substantially similar will preferably haveat least 90% identity (more preferably at least 95% and most preferably99-100%) to the sequence of SEQ ID NO:2.

[0046] The ALP polypeptides of the present invention preferably have asubstantially similar biological activity to the protein encoded by thefull length nucleic acid sequence set forth in SEQ ID NO:1 or to theproteins with amino acid sequence set forth in SEQ ID NO:2. By“biological activity” it is meant an activity of the ALP protein in acell. The biological activity of the ALP is related to some of theactivities of the cell which include, but are not limited to, cellproliferation motogenesis, metastasis, tumor escape, cell adhesion,transformation, or apoptosis.

[0047] By “identity” is meant a property of sequences that measurestheir similarity or relationship. Identity is measured by dividing thenumber of identical residues in the two sequences by the total number ofresidues and multiplying the product by 100. Thus, two copies of exactlythe same sequence have 100% identity, but sequences that are less highlyconserved and have deletions, additions, or replacements have a lowerdegree of identity. Those skilled in the art will recognize that severalcomputer programs are available for determining sequence identity.

[0048] By “isolated” in reference to a polypeptide is meant a polymer of6, 12, 18 or more amino acids conjugated to each other, includingpolypeptides that are isolated from a natural source or that aresynthesized. The isolated polypeptides of the present invention areunique in the sense that they are not found in a pure or separated statein nature. Use of the term “isolated” indicates that a naturallyoccurring sequence has been removed from its normal cellularenvironment. Thus, the sequence may be in a cell-free solution or placedin a different cellular environment. The term does not imply that thesequence is the only amino acid chain present, but that it isessentially free (about 90-95% pure at least) of material naturallyassociated with it.

[0049] By the use of the term “enriched” in reference to a polypeptideit is meant that the specific amino acid sequence constitutes asignificantly higher fraction (2-5 fold) of the total of amino acidsequences present in the cells or solution of interest than in normal ordiseased cells or in the cells from which the sequence was taken. Thiscould be caused by a person by preferential reduction in the amount ofother amino acid sequences present, or by a preferential increase in theamount of the specific amino acid sequence of interest, or by acombination of the two. However, it should be noted that “enriched” doesnot imply that there are no other amino acid sequences present, justthat the relative amount of the sequence of interest has beensignificantly increased.

[0050] The term “significant” here is used to Indicate that the level ofincrease is useful to the person making such an increase, and generallymeans an increase relative to other amino acid sequences of about atleast 2 fold, more preferably at least 5 to 10 fold or even more. Theterm also does not imply that there are no amino acid sequences fromother sources. The other source amino acid may, for example, compriseamino acid sequences encoded by a yeast or bacterial genome, or acloning vector such as pUC19. The term is meant to cover only thosesituations in which a person has intervened to elevate the proportion ofthe desired nucleic acid.

[0051] It is also advantageous for some purposes that an amino acidsequence be in purified form. The term “purified” in reference to apolypeptide does not require absolute purity (such as a homogeneouspreparation); instead, it represents an indication that the sequence isrelatively purer than in the natural environment (compared to thenatural level this level should be at least 2-5 fold greater, e.g., interms of mg/mL). Purification of at least one order of magnitude,preferably two or three orders, and more preferably four or five ordersof magnitude is expressly contemplated. The substance is preferably freeof contamination at a functionally significant level, for example 90%,95%, or 99% pure.

[0052] In another aspect the invention features an isolated, enriched,or purified ALP polypeptide fragment.

[0053] By “an ALP polypeptide fragment” it is meant an amino acidsequence that is less than the full-length ALP amino acid sequence shownin SEQ ID NO:2. Examples of fragments include ALP domains, ALP mutantsand ALP-specific epitopes.

[0054] By “an ALP domain” it is meant a portion of the ALP polypeptidehaving homology to amino acid sequences from one or more known proteinswherein the sequence predicts some common function, interaction oractivity. Well known examples of domains are the SH2 (Src Homology 2)domain (Sadowski, et al., Mol. Cell. Biol. 6:4396, 1986; Pawson andSchlessinger, Curr. Biol. 3:434, 1993), the SH3 domain (Mayer, et al.,Nature 332:272, 1988; Pawson and Schlessinger, Curr. Biol. 3:434, 1993),and pleckstrin (PH) domain (Ponting, TIBS 21:245, 1996; Haslam, et al.,Nature 363:309, 1993), all of which are domains that mediateprotein:protein interaction, and the kinase catalytic domain (Hanks andHunter, FASEB J 9:576-595, 1995). Computer programs designed to detectsuch homologies are well known in the art. The relative homology is atleast 20%, more preferably at least 30% and most preferably at least35%.

[0055] By a “ALP mutant” it is meant an ALP polypeptide which differsfrom the native sequence in that one or more amino acids have beenchanged, added or deleted. Changes in amino acids may be conservative ornon-conservative. By “conservative” it is meant the substitution of anamino acid for one with similar properties such as charge,hydorphobicity, structure, etc. Examples of polypeptides encompased bythis term include, but are not limited to, (1) chimeric proteins whichcomprise a portion of an ALP polypeptide sequence fused to a non-ALPpolypeptide sequence, for example a polypeptide sequence ofhemmaglutinin (HA), (2) ALP proteins lacking a specific domain, forexample the catalytic domain, and (3) ALP proteins having a pointmutation. An ALP mutant will retain some useful function such as, forexample, binding to a natural binding partner, catalytic activity, orthe ability to bind to an ALP specific antibody (as defined below).

[0056] By “ALP-specific epitope” it is meant a sequence of amino acidsthat is both antigenic and unique to ALP. ALP-specific epitope can beused to produce ALP-specific antibodies, as more fully described below.A particularly preferred sequence is amino acids 1 to 352 of SEQ IDNO:2.

[0057] By “recombinant ALP polypeptide” it is meant to include apolypeptide produced by recombinant DNA techniques such that it isdistinct from a naturally occurring polypeptide either in its location(e.g., present in a different cell or tissue than found in nature),purity or structure. Generally, such a recombinant polypeptide will bepresent in a cell in an amount different from that normally observed innature.

[0058] The polypeptide of the invention comprises an amino acid sequencehaving (a) the full length amino acid sequence set forth in SEQ ID NO:2;(b) the full length amino acid sequence of the sequence set forth in SEQID NO:2, except that it lacks one or more of the following segments ofamino acid residues: 1-857, 353-777, 858-1096, 1097-1274, 1101-1214 ofSEQ ID NO:2; (c) the amino acid sequence set forth in SEQ ID NO:2 fromamino acid residues, 1-857, 353-777, 858-1096, 1097-1274, 1101-1214 ofSEQ ID NO:2; or (d) the full length amino acid sequence set forth in SEQID NO:2 except that it lacks one or more of the domains selected fromthe group consisting of an N-terminal domain, an N-terminal proline-richdomain, a catalytic domain, a C-terminal proline/serine-rich domain, anda C-terminal domain.

[0059] In yet another aspect the invention features an antibody (e.g., amonoclonal or polyclonal antibody) having specific binding affinity toan ALP polypeptide or ALP polypeptide fragment. By “specific bindingaffinity” is meant that the antibody binds to target (ALP) polypeptideswith greater affinity than it binds to other polypeptides underspecified conditions. Antibodies having specific binding affinity to anALP polypeptide may be used in methods for detecting the presence and/oramount of an ALP polypeptide in a sample by contacting the sample withthe antibody under conditions such that an immunocomplex forms anddetecting the presence and/or amount of the antibody conjugated to theALP polypeptide. Diagnostic kits for performing such methods may beconstructed to include a first container containing the antibody and asecond container having a conjugate of a binding partner of the antibodyand a label, such as, for example, a radioisotope. The diagnostic kitmay also include notification of an FDA approved use and instructionstherefor.

[0060] The term “polyclonal” refers to antibodies that are heterogenouspopulations of antibody molecules derived from the sera of animalsimmunized with an antigen or an antigenic functional derivative thereof.For the production of polyclonal antibodies, various host animals may beimmunized by injection with the antigen. Various adjuvants may be usedto increase the immunological response, depending on the host species.

[0061] “Monoclonal antibodies” are substantially homogenous populationsof antibodies to a particular antigen. They may be obtained by anytechnique which provides for the production of antibody molecules bycontinuous cell lines in culture. Monoclonal antibodies may be obtainedby methods known to those skilled in the art. See, for example, Kohler,et al., Nature 256:495-497 (1975), and U.S. Pat. No. 4,376,110.

[0062] The term “antibody fragment” refers to a portion of an antibody,often the hypervariable region and portions of the surrounding heavy andlight chains, that displays specific binding affinity for a particularmolecule. A hypervariable region is a portion of an antibody thatphysically binds to the polypeptide target.

[0063] In another aspect the invention features a hybridoma whichproduces an antibody having specific binding affinity to an ALPpolypeptide. By “hybridoma” is meant an immortalized cell line which iscapable of secreting an antibody, for example an ALP antibody. Inpreferred embodiments the ALP antibody comprises a sequence of aminoacids that is able to specifically bind an ALP polypeptide.

[0064] The invention features a method for identifying human cellscontaining an ALP polypeptide or a related sequence. The method involvesidentifying the novel polypeptide in human cells using techniques thatare routine and standard in the art, such as those described herein foridentifying ALP (e.g., cloning, Southern or Northern blot analysis, insitu hybridization, PCR amplification, etc.).

[0065] The invention also features methods of screening cells fornatural binding partners of ALP polypeptides. By “natural bindingpartner” it is meant a protein that interacts with ALP. Binding partnersinclude ligands, agonists, antagonists and downstream signalingmolecules such as adaptor proteins and may be identified by techniqueswell known in the art such as co-immunoprecipitation or by using, forexample, a two-hybrid screen. (Fields and Song, U.S. Pat. No. 5,283,173,issued Feb. 1, 1994 and, incorporated be reference herein.) The presentinvention also features the purified, isolated or enriched versions ofthe polypeptides identified by the methods described above.

[0066] In another aspect, the invention provides a method foridentifying a substance capable of modulating ALP activity comprisingthe steps of (a) contacting an ALP polypeptide with a test substance;and (b) determining whether the substance alters the activity of saidpolypeptide.

[0067] The invention also features another method of identifyingsubstances capable of modulating the function of a polypeptide. Themethod comprises the following steps: (a) expressing an ALP polypeptidein cells; (b) adding a compound to the cells; and (c) monitoring achange or an absence of a change in cell phenotype, cell proliferation,catalytic activity of the ALP polypeptide, and binding a natural bindingpartner.

[0068] The term “compound” includes small organic molecules including,but not limited to, oxindolinones, quinazolines, tyrphostins,quinoxalines, and those contained within extracts from natural sources.Examples of such compounds are included in section XII, below.

[0069] The term “function” refers to the cellular role of aserine-threonine protein kinase. The serine-threonine protein kinasefamily includes members that regulate many steps in signaling cascades,including cascades controlling cell growth, migration, differentiation,gene expression, muscle contraction, glucose metabolism, cellularprotein synthesis, and regulation of the cell cycle.

[0070] The term “modulates” refers to the ability of a compound to alterthe function of a protein kinase. A modulator preferably activates thecatalytic activity of a protein kinase, more preferably activates orinhibits the catalytic activity of a protein kinase depending on theconcentration of the compound exposed to the protein kinase, or mostpreferably inhibits the catalytic activity of a protein kinase.

[0071] The term “catalytic activity,” in the context of the invention,defines the ability of a protein kinase to phosphorylate a substrate.Catalytic activity can be measured, for example, by determining theamount of a substrate converted to a product as a function of time.Phosphorylation of a substrate occurs at the active-site of a proteinkinase. The active-site is normally a cavity in which the substrate.

[0072] The term “substrate” as used herein refers to a molecule that isphoshorylated by or directly interacts with the protein kinase. Thesubstrate is preferably a peptide and more preferably a protein. Forexample, in relation to the protein kinase RAF, preferred substrates areMEK and the MEK substrate MAPK.

[0073] The term “activates” refers to increasing the cellular functionof a protein kinase. The protein kinase function is preferably theinteraction with a natural binding partner or catalytic activity. Theterm “inhibit” refers to decreasing the cellular function of a proteinkinase. The protein kinase function is preferably the interaction with anatural binding partner or catalytic activity.

[0074] The term “modulates” also refers to altering the function of aprotein kinase by increasing or decreasing the probability that acomplex forms between a protein kinase and a natural binding partner. Amodulator preferably increases the probability that such a complex formsbetween the protein kinase and the natural binding partner, morepreferably increases or decreases the probability that a complex formsbetween the protein kinase and the natural binding partner depending onthe concentration of the compound exposed to the protein kinase, andmost preferably decreases the probability that a complex forms betweenthe protein kinase and the natural binding partner.

[0075] The term “complex” refers to an assembly of at least twomolecules bound to one another. Signal transduction complexes oftencontain at least two protein molecules bound to one another, eithertransiently or in succession. For instance, a receptor protein tyrosinekinase, GRB2, SOS, and RAF sequentially interact in response to amitogenic ligand.

[0076] The term “expressing” as used herein refers to the production ofan ALP polypeptide from a nucleic acid vector containing an ALP genewithin a cell. The nucleic acid vector is transfected into cells usingwell known techniques in the art as described herein.

[0077] The term “adding” as used herein refers to administering asolution comprising a compound to the medium bathing cells. The solutioncomprising the compound can also comprise an agent, such as dimethylsulfoxide, which facilitates the uptake of the compound into the cells.

[0078] The term “monitoring” refers to observing the effect of addingthe compound to the cells of the method. The effect can be manifested ina change in cell phenotype, cell proliferation, protein kinase catalyticactivity, or in the interaction between a protein kinase and a naturalbinding partner.

[0079] The term “cell phenotype” refers to the outward appearance of acell or tissue or the function of the cell or tissue. Examples of cellor tissue phenotype are cell size (reduction or enlargement), cellproliferation (increased or decreased numbers of cells), celldifferentiation (a change or absence of a change in cell shape), cellsurvival, apoptosis (cell death), or the utilization of a metabolicnutrient (e.g., glucose uptake). Change or the absence of change in cellphenotype is readily measured by techniques known in the art.

[0080] The term “cell proliferation” refers to the rate at which a groupof cells divides. The number of cells growing in a vessel can bequantitated by a person skilled in the art when that person visuallycounts the number of cells in a defined area using a common lightmicroscope. Alternatively, cell proliferation rates can be quantitatedby laboratory apparatae that optically measure the density of cells inan appropriate medium.

[0081] The method can utilize any of the molecules disclosed in theinvention. These molecules include nucleic acid molecules encoding ALPpolypeptides, nucleic acid vectors, recombinant cells, polypeptides, orantibodies of the invention.

[0082] In a preferred embodiment, the invention provides a method fortreating or preventing an abnormal condition by administering a compoundwhich is a modular of ALP function in vitro. The abnormal conditionpreferably involves abnormality in ALP signal transduction pathway, andmost preferably is cancer. Such compounds preferably show positiveresults in one or more in vitro assays for an activity corresponding totreatment of the disease or disorder in question (such as the assaysdescribed in Example 6 below). Examples of substances that can bescreened for favorable activity are provided in section XII below.

[0083] The summary of the invention described above is non-limiting andother features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DETAILED DESCRIPTION OF THE INVENTION

[0084] The present invention relates to the isolation andcharacterization of a new protein which we have called ALP, nucleotidesequences encoding ALP, various products and assay methods that can beused to identify compounds useful for the diagnosis and treatment ofvarious ALP related diseases and conditions, for example cancer.Polypeptides derived from ALP and nucleic acids encoding suchpolypeptides may be produced using well known and standard synthesistechniques when given the sequences presented herein.

[0085] ALP is a tyrosine phosphatase with an apparent molecular weightof approximately 160-200 kDa. Primary sequence analysis shows that ALPis comprised of three domains: a domain at the N-terminus that is richin proline residues (30.6%) and contains several tyrosines that may bephosphorylated, a catalytic domain, and a C-terminal domain containingregion rich in prolines and serines (45.6%) that resenbling a PEST motif(Rogers, et al; Science 234:364, 1986). These proline rich regions maybe protein:protein interaction sites as SH3 domains have been shown tobind to proline rich regions (Morton and Campbell, Curr. Biol. 4:614,1994; Ren, et al; Science 259:1157, 1993). The lack of a hydrophobicstretch of amino acids generally characterized as a transmembrane regionindicates that ALP is a non-receptor tyrosine phosphatase.

[0086] The full-length ALP was originally isolated from a human braincancer cell line. Subsequent expression analysis of both normal tissuesand cancer cell lines, shown in detail below, revealed that ALP has lowexpression in normal cells but is significantly overexpressed in anumber of tumors. This suggests that ALP plays an important role in thegrowth and persistance of these cancers.

[0087] The polypeptide and nucleotide sequences of the invention can beused, therefore, to identify modulators of cell growth and survivalwhich are useful in developing therapeutics for various cellproliferative disorders and conditions, and in particular cancersrelated to inappropriate ALP activity. Assays to identify compounds thatact intracellularly to enhance or inhibit ALP activity can be developedby creating genetically engineered cell lines that express ALPnucleotide sequences, as is more fully discussed below.

[0088] I. Nucleic Acids Encoding ALP Polypeptides

[0089] A first aspect of the invention features nucleic acid sequencesencoding an ALP polypeptide. Included within the scope of this inventionare the functional equivalents of the herein-described isolated nucleicacid molecules. Functional equivalents or derivatives can be obtained inseveral ways. The degeneracy of the genetic code permits substitution ofcertain codons by other codons which specify the same amino acid andhence would give rise to the same protein. The nucleic acid sequence canvary substantially since, with the exception of methionine andtryptophan, the known amino acids can be coded for by more than onecodon. Thus, portions or all of the ALP gcne could be synthesized togive a nucleic acid sequence significantly different from that shown inSEQ ID NO:1. The encoded amino acid sequence thereof would, however, bepreserved.

[0090] In addition, the nucleic acid sequence may comprise a nucleotidesequence which results from the addition, deletion or substitution of atleast one nucleotide to the 5′-end and/or the 3′-end of the nucleic acidformula shown in SEQ ID NO:1 or a derivative thereof. Any nucleotide orpolynucleotide may be used in this regard, provided that its addition,deletion or substitution does not alter the amino acid sequence of SEQID NO:2 which is encoded by the nucleotide sequence. For example, thepresent invention is intended to include any nucleic acid sequenceresulting from the addition of ATG as an initiation codon at the 5′-endof the ALP nucleic acid sequence or its functional derivative, or fromthe addition of TTA, TAG or TGA as a termination codon at the 3′-end ofthe inventive nucleotide sequence or its derivative. Moreover, thenucleic acid molecule of the present invention may, as necessary, haverestriction endonuclease recognition sites added to its 5′-end and/or3′-end.

[0091] Such functional alterations of a given nucleic acid sequenceafford an opportunity to promote secretion and/or processing ofheterologous proteins encoded by foreign nucleic acid sequences fusedthereto. All variations of the nucleotide sequence of the ALP genes andfragments thereof permitted by the genetic code are, therefore, includedin this invention.

[0092] Further, it is possible to delete codons or to substitute one ormore codons by codons other than degenerate codons to produce astructurally modified polypeptide, but one which has substantially thesame utility or activity of the polypeptide produced by the unmodifiednucleic acid molecule. As recognized in the art, the two polypeptidesare functionally equivalent, as are the two nucleic acid molecules whichgive rise to their production, even though the differences between thenucleic acid molecules are not related to degeneracy of the geneticcode.

[0093] Functional equivalents or derivatives of ALP can also be obtainedusing nucleic acid molecules encoding one or more functional domains ofthe ALP polypeptide. For example, the N-terminal proline-rich domain ofALP functions as a SH3 binding domain and a nucleic acid sequenceencoding the N-terminal proline-rich domain alone or linked to otherheterologous nucleic acid sequences can be considered a functionalderivative of ALP. Other functional domains of ALP include, but are notlimited to, the proline-rich region within the N-terminal proline-richdomain, the C-terminal proline/serine-rich domain, theproline/serine-rich region within the C-terminal proline/serin-richdomain, and the catalytic domain. Nucleic acid sequences encoding thesedomains are shown in SEQ ID NO:1 as follows: N-terminal domain 313-2883;proline-rich region 1369-2643; catalytic domain approximately 2884-3600,C-terminal proline/serine-rich domain 3601-4134, proline/serine-richregion 3613-4456.

[0094] II. A Nucleic Acid Probe for the Detection of ALP

[0095] A nucleic acid probe of the present invention may be used toprobe an appropriate chromosomal or cDNA library by usual hybridizationmethods to obtain another nucleic acid molecule of the presentinvention. A chromosomal DNA or cDNA library may be prepared fromappropriate cells according to recognized methods in the art (e.g.“Molecular Cloning: A Laboratory Manual”, second edition, edited bySambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989).

[0096] In the alternative, chemical synthesis is carried out in order toobtain nucleic acid probes having nucleotide sequences which correspondto N-terminal and C-terminal portions of the amino acid sequence of thepolypeptide of interest. Thus, the synthesized nucleic acid probes maybe used as primers in a polymerase chain reaction (PCR) carried out inaccordance with recognized PCR techniques, essentially according to PCRProtocols, “PCR Protocols, A Guide to Methods and Applications”, editedby Innis et al., Academic Press, 1990, utilizing the appropriatechromosomal or cDNA library to obtain the fragment of the presentnvention.

[0097] One skilled in the art can readily design such probes based onthe sequence disclosed herein using methods of computer alignment andsequence analysis known in the art (e.g. “Molecular Cloning: ALaboratory Manual”, second edition, edited by Sambrook, Fritsch, &Maniatis, Cold Spring Harbor Laboratory, 1989). The hybridization probesof the present invention can be labeled by standard labeling techniquessuch as with a radiolabel, enzyme label, fluorescent label,biotin-avidin label, chemiluminescence, and the like. Afterhybridization, the probes may be visualized using known methods.

[0098] The nucleic acid probes of the present invention include RNA aswell as DNA probes and nucleic acids modified in the sugar, phosphate oreven the base portion as long as the probe still retains the ability tospecifically hybridize under conditions as disclosed herein. Such probesare generated using techniques known in the art. The nucleic acid probemay be immobilized on a solid support. Examples of such solid supportsinclude, but are not limited to, plastics such as polycarbonate, complexcarbohydrates such as agarose and sepharose, acrylic resins, such aspolyacrylamide and latex beads, and nitrocellulose. Techniques forcoupling nucleic acid probes to such solid supports are well known inthe art.

[0099] The test samples suitable for nucleic acid probing methods of thepresent invention include, for example, cells or nucleic acid extractsof cells, or biological fluids. The sample used in the above-describedmethods will vary based on the assay format, the detection method andthe nature of the tissues, cells or extracts to be assayed. Methods forpreparing nucleic acid extracts of cells are well known in the art andcan be readily adapted in order to obtain a sample which is compatiblewith the method utilized.

[0100] III. A Probe Based Method And Kit For Detecting ALP

[0101] One method of detecting the presence of ALP in a sample comprises(a) contacting the sample with the above-described nucleic acid probe,under conditions such that hybridization occurs, and (b) detecting thepresence of the probe bound to a nucleic acid molecule in the sample.One skilled in the art would select the nucleic acid probe according totechniques known in the art as described above. Samples to be testedinclude but should not be limited to RNA samples of human tissue.

[0102] A kit for detecting the presence of ALP in a sample comprises atleast one container having disposed therein an above-described nucleicacid probe. The kit may further comprise other containers comprising oneor more of the following: wash reagents and reagents capable ofdetecting the presence of bound nucleic acid probe. Examples ofdetection reagents include, but are not limited to radiolabelled probes,enzymaticly labeled probes (horseradish peroxidase, alkalinephosphatase), and affinity labeled probes (biotin, avidin, orsteptavidin).

[0103] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers or strips of plastic orpaper. Such containers allow the efficient transfer of reagents from onecompartment to another compartment such that the samples and reagentsare not cross-contaminated and the agents or solutions of each containercan be added in a quantitative fashion from one compartment to another.Such containers will include a container which will accept the testsample, a container which contains the probe or primers used in theassay, containers which contain wash reagents (such as phosphatebuffered saline, Tris-buffers, and the like), and containers whichcontain the reagents used to detect the hybridized probe, boundantibody, amplified product, or the like. One skilled in the art willreadily recognize that the nucleic acid probes described in the presentinvention can readily be incorporated into one of the established kitformats which are well known in the art.

[0104] IV. DNA Constructs Comprising an ALP Nucleic Acid Molecule andCells Containing These Constructs

[0105] The present invention also relates to a recombinant DNA moleculecomprising, 5′ to 3′, a promoter effective to initiate transcription ina host cell and one of above-described nucleic acid molecules. Inaddition, the present invention relates to a recombinant DNA moleculecomprising a vector and a nucleic acid molecule described herein. Thepresent invention also relates to a nucleic acid molecule comprising atranscriptional region functional in a cell, a sequence complimentary toan RNA sequence encoding an amino acid sequence corresponding to an ALPpolypeptide or functional derivative, and a transcriptional terminationregion functional in said cell. The above-described molecules may beisolated and/or purified DNA molecules.

[0106] The present invention also relates to a cell or organism thatcontains an ALP nucleic acid molecule as described herein and thereby iscapable of expressing a peptide. The polypeptide may be purified fromcells which have been altered to express the polypeptide. A cell is saidto be “altered to express a desired polypeptide” when the cell, throughgenetic manipulation, is made to produce a protein which it normallydoes not produce or which the cell normally produces at lower levels.One skilled in the art can readily adapt procedures for introducing andexpressing either genomic, cDNA, or synthetic sequences into eithereukaryotic or prokaryotic cells.

[0107] A nucleic acid molecule, such as DNA, is said to be “capable ofexpressing” a polypeptide if it contains nucleotide sequences whichcontain transcriptional and translational regulatory information andsuch sequences are “operably linked” to nucleotide sequences whichencode the polypeptide. An operable linkage is a linkage in which theregulatory DNA sequences and the DNA sequence sought to be expressed areconnected in such a way as to permit gene sequence expression. Theprecise nature of the regulatory regions needed for gene sequenceexpression may vary from organism to organism, but will in generalinclude a promoter region which, in prokaryotes, contains both thepromoter (which directs the initiation of RNA transcription) as well asthe DNA sequences which, when transcribed into RNA, will signalsynthesis initiation. Such regions will normally include those5′-non-coding sequences involved with initiation of transcription andtranslation, such as the TATA box, capping sequence, CAAT sequence, andthe like.

[0108] If desired, the non-coding region 3′ to the sequence encoding anALP gene may be obtained by the above-described cloning methods. Thisregion may be retained for its transcriptional termination regulatorysequences, such as termination and polyadenylation. Thus, by retainingthe 3′-region naturally contiguous to the DNA sequence encoding an ALPgene, the transcriptional termination signals may be provided. Where thetranscriptional termination signals are not satisfactorily functional inthe expression host cell, then a 3′ region functional in the host cellmay be substituted.

[0109] Two DNA sequences (such as a promoter region sequence and an ALPsequence) are said to be operably linked if the nature of the linkagebetween the two DNA sequences does not (1) result in the introduction ofa frame-shift mutation, (2) interfere with the ability of the promoterregion sequence to direct the transcription of the second sequence, forexample an ALP gene sequence, or (3) interfere with the ability of thesecond sequence to be transcribed by the promoter region sequence. Thus,a promoter region would be operably linked to a DNA sequence if thepromoter were capable of effecting transcription of that DNA sequence.Thus transcriptional and translational signals recognized by anappropriate host are necessary to express an ALP gene.

[0110] The present invention encompasses the expression of an ALP gene(or a functional derivative thereof) in either prokaryotic or eukaryoticcells. Prokaryotic hosts are, generally, very efficient and convenientfor the production of recombinant proteins and are, therefore, one typeof preferred expression system for an ALP gene. Prokaryotes mostfrequently are represented by various strains of E. coli. However, othermicrobial strains may also be used, including other bacterial strains.

[0111] In prokaryotic systems, plasmid vectors that contain replicationsites and control sequences derived from a species compatible with thehost may be used. Examples of suitable plasmid vectors may includepBR322, pUC118, pUC119 and the like; suitable phage or bacteriophagevectors may include λgt10, λgt11 and the like; and suitable virusvectors may include pMAM-neo, pKRC and the like. Preferably, theselected vector of the present invention has the capacity to replicatein the selected host cell.

[0112] Recognized prokaryotic hosts include bacteria such as E. coli andthose from genera such as Bacillus, Streptomyces, Pseudomonas,Salmonella, Serratia, and the like. However, under such conditions, thepolypeptide will not be glycosylated. The prokaryotic host must becompatible with the replicon and control sequences in the expressionplasmid.

[0113] To express ALP (or a functional derivative thereof) in aprokaryotic cell, it is necessary to operably link an ALP sequence to afunctional prokaryotic promoter. Such promoters may be eitherconstitutive or, more preferably, regulatable (i.e., inducible orderepressible). Examples of constitutive promoters include the intpromoter of bacteriophage λ, the bla promoter of the β-lactamase genesequence of pBR322, and the CAT promoter of the chloramphenicol acetyltransferase gene sequence of pPR325, and the like. Examples ofconstitutive promoters include the int promoter of bacteriophage 1, thebla promoter of the b-lactamase gene sequence of pBR322, and the CATpromoter of the chloramphenicol acetyl transferase gene sequence ofpPR325, and the like. Examples of inducible prokaryotic promotersinclude the major right and left promoters of bacteriophage l (P_(L) andP_(R)), the trp, recA, lacZ, laci, and gal promoters of E. coli, thea-amylase (Ulmanen et al., J. Bacteriol. 162:176-182, 1985) and thesigma-28-specific promoters of B. subtilis (Gilman et al., Gene Sequence32:11-20(1984)), the promoters of the bacteriophages of Bacillus(Gryczan, In: The Molecular Biology of the Bacilli, Academic Press,Inc., NY (1982)), and Streptomyces promoters (Ward et al., Mol. Gen.Genet. 203:468-478, 1986). Prokaryotic promoters are reviewed by Glick(J. Ind. Microbiot. 1:277-282, 1987); Cenatiempo (Biochimie 68:505-516,1986); and Gottesman (Ann. Rev. Genet. 18:415-442, 1984).

[0114] Proper expression in a prokaryotic cell also requires thepresence of a ribosome binding site upstream of the genesequence-encoding sequence. Such ribosome binding sites are disclosed,for example, by Gold et at. (Ann. Rev. Microbiol. 35:365-404, 1981). Theselection of control sequences, expression vectors, transformationmethods, and the like, are dependent on the type of host cell used toexpress the gene.

[0115] As used herein, “cell”, “cell line”, and “cell culture” may beused interchangeably and all such designations include progeny. Thus,the words “transformants” or “transformed cells” include the primarysubject cell and cultures derived therefrom, without regard to thenumber of transfers. It is also understood that all progeny may not beprecisely identical in DNA content, due to deliberate or inadvertentmutations. However, as defined, mutant progeny have the samefunctionality as that of the originally transformed cell.

[0116] Host cells which may be used in the expression systems of thepresent invention are not strictly limited, provided that they aresuitable for use in the expression of the ALP peptide of interest.Suitable hosts may often include eukaryotic cells. Preferred eukaryotichosts include, for example, yeast, fungi, insect cells, and mammaliancells, either in vivo or in tissue culture. Mammalian cells which may beuseful as hosts include HeLa cells, cells of fibroblast origin such asVERO, 3T3 or CHO-K1, or cells of lymphoid origin (such as 32D cells) andtheir derivatives. Preferred mammalian host cells include SP2/0 andJ558L, as well as neuroblastoma cell lines such as IMR 332 and PC12which may provide better capacities for correct post-translationalprocessing.

[0117] In addition, plant cells are also available as hosts, and controlsequences compatible with plant cells are available, such as thecauliflower mosaic virus 35S and 19S, and nopaline synthase promoter andpolyadenylation signal sequences. Another preferred host is an insectcell, for example the Drosophila larvae. Using insect cells as hosts,the Drosophila alcohol dehydrogenase promoter can be used. Rubin,Science 240:1453-1459, 1988). Alternatively, baculovirus vectors can beengineered to express large amounts of ALP in insects cells (Jasny,Science 238:1653, 1987); Miller et al., In: Genetic Engineering (1986),Setlow, J. K., et al., eds., Plenum, Vol. 8, pp. 277-297).

[0118] Any of a series of yeast gene sequence expression systems can beutilized which incorporate promoter and termination elements from theactively expressed gene sequences coding for glycolytic enzymes areproduced in large quantities when yeast are grown in mediums rich inglucose. Known glycolytic gene sequences can also provide very efficienttranscriptional control signals. Yeast provides substantial advantagesin that it can also carry out post-translational peptide modifications.A number of recombinant DNA strategies exist which utilize strongpromoter sequences and high copy number of plasmids which can beutilized for production of the desired proteins in yeast. Yeastrecognizes leader sequences on cloned mammalian gene sequence productsand secretes peptides bearing leader sequences (i.e., pre-peptides). Fora mammalian host, several possible vector systems are available for theexpression of ALP.

[0119] A particularly preferred yeast expression system is thatutilizing Schizosaccharmocyces pombe. This system is useful for studyingthe activity of members of the Src family (Superti-Furga, et al, EMBO J.12:2625, 1993) and other non-receptor tyrosine kinases, the function ofwhich is often regulated by the activity of tyrosine phosphatases.

[0120] A wide variety of transcriptional and translational regulatorysequences may be employed, depending upon the nature of the host. Thetranscriptional and translational regulatory signals may be derived fromviral sources, such as adenovirus, bovine papilloma virus,cytomegalovirus, simian virus, or the like, where the regulatory signalsare associated with a particular gene sequence which has a high level ofexpression. Alternatively, promoters from mammalian expression products,such as actin, collagen, myosin, and the like, may be employed.Transcriptional initiation regulatory signals may be selected whichallow for repression or activation, so that expression of the genesequences can be modulated. Of interest are regulatory signals which aretemperature-sensitive so that by varying the temperature, expression canbe repressed or initiated, or are subject to chemical (such asmetabolite) regulation.

[0121] Expression of ALP in eukaryotic hosts requires the use ofeukaryotic regulatory regions. Such regions will, in general, include apromoter region sufficient to direct the initiation of RNA synthesis.Preferred eukaryotic promoters include, for example, the promoter of themouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen.1:273-288, 1982); the TK promoter of Herpes virus (McKnight, Cell31:355-365, 1982); the SV40 early promoter (Benoist et al., Nature(London) 290:304-310, 1981); the yeast gal4 gene sequence promoter(Johnston et al., Proc. Natl. Acad. Sci. USA 79:6971-6975, 1982); Silveret al., Proc. Natl. Acad. Sci. USA 81:5951-5955, 1984).

[0122] Translation of eukaryotic mRNA is initiated at the codon whichencodes the first methionine. For this reason, it is preferable toensure that the linkage between a eukaryotic promoter and a DNA sequencewhich encodes ALP (or a functional derivative thereof) does not containany intervening codons which are capable of encoding a methionine (i.e.,AUG). The presence of such codons results either in the formation of afusion protein (if the AUG codon is in the same reading frame as the ALPcoding sequence) or a frame-shift mutation (if the AUG codon is not inthe same reading frame as the ALP coding sequence).

[0123] An ALP nucleic acid molecule and an operably linked promoter maybe introduced into a recipient prokaryotic or eukaryotic cell either asa nonreplicating DNA (or RNA) molecule, which may either be a linearmolecule or, more preferably, a closed covalent circular molecule (aplasmid). Since such molecules are incapable of autonomous replication,the expression of the gene may occur through the transient expression ofthe introduced sequence. Alternatively, permanent or stable expressionmay occur through the integration of the introduced DNA sequence intothe host chromosome.

[0124] A vector may be employed which is capable of integrating thedesired gene sequences into the host cell chromosome. Cells which havestably integrated the introduced DNA into their chromosomes can beselected by also introducing one or more markers which allow forselection of host cells which contain the expression vector. The markermay provide for prototrophy to an auxotrophic host, biocide resistance,e.g., antibiotics, or heavy metals, such as copper, or the like. Theselectable marker gene sequence can either be directly linked to the DNAgene sequences to be expressed, or introduced into the same cell byco-transfection. Additional elements may also be needed for optimalsynthesis of single chain binding protein mRNA. These elements mayinclude splice signals, as well as transcription promoters, enhancers,and termination signals. cDNA expression vectors incorporating suchelements include those described by Okayama, Mol. Cell. Bio. 3:280,1983.

[0125] The introduced nucleic acid molecule can be incorporated into aplasmid or viral vector capable of autonomous replication in therecipient host. Any of a wide variety of vectors may be employed forthis purpose. Factors of importance in selecting a particular plasmid orviral vector include: the ease with which recipient cells that containthe vector may be recognized and selected from those recipient cellswhich do not contain the vector; the number of copies of the vectorwhich are desired in a particular host; and whether it is desirable tobe able to “shuttle” the vector between host cells of different species.

[0126] Preferred prokaryotic vectors include plasmids such as thosecapable of replication in E. coil (such as, for example, pBR322, ColEl,pSC101, pACYC 184, pVX. Such plasmids are, for example, disclosed bySambrook (cf. “Molecular Cloning: A Laboratory Manual”, second edition,edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory,(1989)). Bacillus plasmids include pC194, pC221, pTl27, and the like.Such plasmids are disclosed by Gryczan (In: The Molecular Biology of theBacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomycesplasmids include plJ101 (Kendall et al., J. Bacteriol.169:4177-4183,1987), and streptomyces bacteriophages such as fC31(Chater et al., In: Sixth International Symposium on ActinomycetalesBiology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54).Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis.8:693-704, 1986), and Izaki (Jpn. J. Bacteriol. 33:729-742, 1978).

[0127] Preferred eukaryotic plasmids include, for example, BPV,vaccinia, SV40, 2-micron circle, and the like, or their derivatives.Such plasmids are well known in the art (Botstein et al., Miami Wntr.Symp. 19:265-274, 1982); Broach, In: “The Molecular Biology of the YeastSaccharomyces: Life Cycle and Inheritance”, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., p. 445-470 (1981); Broach, Cell28:203-204, 1982); Bollon et at., J. Clin. Hematol. Oncol. 10:39-48,1980); Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3,Gene Sequence Expression, Academic Press, NY, pp. 563-608 (1980).

[0128] Once the vector or nucleic acid molecule containing theconstruct(s) has been prepared for expression, the DNA construct(s) maybe introduced into an appropriate host cell by any of a variety ofsuitable means, i.e., transformation, transfection, conjugation,protoplast fusion, electroporation, particle gun technology, calciumphosphate-precipitation, direct microinjection, and the like. After theintroduction of the vector, recipient cells are grown in a selectivemedium, which selects for the growth of vector-containing cells.Expression of the cloned gene molecule(s) results in the production ofALP or fragments or functional derivatives thereof. This can take placein the transformed cells as such, or following the induction of thesecells to differentiate (for example, by administration ofbromodeoxyuracil to neuroblastoma cells or the like). A variety ofincubation conditions for the transformed cells can be used to fosterexpression of the polypeptides of the peptide of the present invention.The most preferred conditions are those which mimic physiologicalconditions.

[0129] V. ALP Polypeptides

[0130] Also a feature of the invention are ALP polypeptides. A varietyof methodologies known in the art can be utilized to obtain thepolypeptides of the present invention. They may be purified from tissuesor cells which naturally produce them. Alternatively, theabove-described isolated nucleic acid sequences can be used to expressan ALP protein recombinantly.

[0131] Any eukaryotic organism can be used as a source for thepolypeptide of the invention, as long as the source organism naturallycontains such a polypeptide. As used herein, “source organism” refers tothe original organism from which the amino acid sequence is derived,regardless of the organism the protein is expressed in and ultimatelyisolated from.

[0132] One skilled in the art can readily follow known methods forisolating proteins in order to obtain the peptide free of naturalcontaminants. These include, but are not limited to: size-exclusionchromatography, HPLC, ion-exchange chromatography, and immuno-affinitychromatography.

[0133] An ALP protein, like all proteins, is comprised of distinctfunctional units or domains. In eukaryotes, proteins sorted through theso-called vesicular pathway (bulk flow) usually have a signal sequence(also called a leader peptide) in the N-terminus, which is cleaved offafter the translocation through the ER (endoplasmic reticulum) membrane.Some N-terminal signal sequences are not cleaved off, remaining astransmembrane segments, but it does not mean these proteins are retainedin the ER; they can be further sorted and included in vesicles.Non-receptor proteins generally function to transmit signals within thecell, either by providing sites for protein:protein interactions or byhaving some catalytic activity (contained within a catalytic domain),often both. Methods of predicting the existence of these various domainsare well known in the art. Protein:protein interaction domains can beidentified by comparison to other proteins. The SH2 domain, for exampleis a protein domain of about 100 amino acids first identified as aconserved sequence region between the proteins Src and Fps (Sadowski; etal, Mol. Cell. Bio. 6:4396, 1986). Similar sequences were later found inmany other intracellular signal-transducing proteins. SH2 domainsfunction as regulatory modules of intracellular signalling cascades byinteracting with high affinity to phosphotyrosine-containing proteins ina sequence specific and strictly phosphorylation-dependent manner (Mayerand Baltimore, Trends Cell. Biol. 3:8, 1993). Kinase or phosphatasecatalytic domains can be identified by comparison to other knowncatalytic domains with kinase phosphatase activity. See, for exampleHanks and Hunter, FASEB J. 9:576-595, 1995.

[0134] Primary sequence analysis of the ALP amino acid sequence (shownin SEQ ID NO: 2) reveals that it does not contain a signal sequence ortransmembrane domain and is, therefore, an intracellular protein.Comparison to known protein sequences revels that ALP is comprised ofseveral unique domains. These include a 857 amino acid N-terminalproline-rich domain (shown from amino acid number 1-857 of SEQ ID NO: 2)within which is a proline-rich region (amino acid number 353-777 of SEQID NO:2), a 238 amino acid catalytic domain (shown from amino acidnumber 858-1096 of SEQ ID NO: 2), and a 177 amino acid C-terminalproline/serine-rich domain (shown from amino acid number 1097-1274 ofSEQ ID NO:2) within which is a proline/serine-rich region (amino acidnumber 1101-1214 of SEQ ID NO:2).

[0135] These ALP domains have a variety of uses. An example of such ause is to make a polypeptide consisting of the ALP catalytic domain anda heterologous protein such as glutathione S-transferase (GST). Such apolypeptide can be used in a biochemical assay for ALP catalyticactivity useful for studying ALP substrate specificity or foridentifying substances that can modulate ALP catalytic activity.Alternatively, one skilled in the art could create an ALP polypetidelacking at least one of the three major domains. Such a polypeptide,when expressed in a cell, is able to form complexes with the natuaralbinding partner(s) of ALP but unable to transmit any signal furtherdownstream into the cell, ie. it would be signaling incompetent and thuswould be useful for studying the biological relevance of ALP activity.(See, as an example, Gishizky, et al; PNAS:10889, 1995).

[0136] VI. An Antibody Having Binding Affinity to an ALP Polypeptide andA Hybridoma Containing the Antibody

[0137] The present invention also relates to an antibody having specificbinding affinity to an ALP polypeptide. The polypeptide may have theamino acid sequence set forth in SEQ ID NO:2, or a be fragment thereof,or at least 6 contiguous amino acids thereof. Such an antibody may beidentified by comparing its binding affinity to an ALP polypeptide withits binding affinity to another polypeptide. Those which bindselectively to ALP would be chosen for use in methods requiring adistinction between ALP and other polypeptides. Such methods couldinclude, but should not be limited to, the analysis of altered ALPexpression in tissue containing other polypeptides and assay systemsusing whole cells.

[0138] An ALP peptide of the present invention can be used to produceantibodies or hybridomas. One skilled in the art will recognize that ifan antibody is desired, such a peptide would be generated as describedherein and used as an immunogen. A preferred ALP peptide in this respectis the sequence from amino acids 1 to 352 of SEQ ID NO:2. The antibodiesof the present invention include monoclonal and polyclonal antibodies,as well fragments of these antibodies, and humanized forms. Humanizedforms of the antibodies of the present invention may be generated usingone of the procedures known in the art such as chimerization or CDRgrafting. The present invention also relates to a hybridoma whichproduces the above-described monoclonal antibody, or binding fragmentthereof. A hybridoma is an immortalized cell line which is capable ofsecreting a specific monoclonal antibody.

[0139] In general, techniques for preparing monoclonal antibodies andhybridomas are well known in the art (Campbell, “Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology,” Elsevier Science Publishers, Amsterdam, The Netherlands, 1984;St. Groth et al., J. Immunol. Methods 35:1-21, 1980). Any animal (mouse,rabbit, and the like) which is known to produce antibodies can beimmunized with the selected polypeptide. Methods for immunization arewell known in the art. Such methods include subcutaneous orintraperitoneal injection of the polypeptide. One skilled in the artwill recognize that the amount of polypeptide used for immunization willvary based on the animal which is immunized, the antigenicity of thepolypeptide and the site of injection.

[0140] The polypeptide may be modified or administered in an adjuvant inorder to increase the peptide antigenicity. Methods of increasing theantigenicity of a polypeptide are well known in the art. Such proceduresinclude coupling the antigen with a heterologous protein (such asglobulin or β-galactosidase) or through the inclusion of an adjuvantduring immunization.

[0141] For monoclonal antibodies, spleen cells from the immunizedanimals are removed, fused with myeloma cells, such as SP2/0-Agl4myeloma cells, and allowed to become monoclonal antibody producinghybridoma cells. Any one of a number of methods well known in the artcan be used to identify the hybridoma cell which produces an antibodywith the desired characteristics. These include screening the hybridomaswith an ELISA assay, western blot analysis, or radioimmunoassay (Lutz,et al., Exp. Cell Res. 175:109-124, 1988). Hybridomas secreting thedesired antibodies are cloned and the class and subclass is determinedusing procedures known in the art (Campbell, “Monoclonal AntibodyTechnology: Laboratory Techniques in Biochemistry and MolecularBiology”, supra, 1984).

[0142] For polyclonal antibodies, antibody containing antisera isisolated from the immunized animal and is screened for the presence ofantibodies with the desired specificity using one of the above-describedprocedures. The above-described antibodies may be detectably labeled.Antibodies can be detectably labeled through the use of radioisotopes,affinity labels (such as biotin, avidin, and the like), enzymatic labels(such as horse radish peroxidase, alkaline phosphatase, and the like)fluorescent labels (such as FITC or rhodamine, and the like),paramagnetic atoms, and the like. Procedures for accomplishing suchlabeling are well-known in the art, for example, see (Stemberger, etal., J. Histochem. Cytochem. 18:315, 1970; Bayer, et at., Meth. Enzym.62:308, 1979; Engval, et al., Immunot. 109:129, 1972; Goding, J.Immunol. Meth. 13:215, 1976). The labeled antibodies of the presentinvention can be used for in vitro, in vivo, and in situ assays toidentify cells or tissues which express a specific peptide.

[0143] The above-described antibodies may also be immobilized on a solidsupport. Examples of such solid supports include plastics such aspolycarbonate, complex carbohydrates such as agarose and sepharose,acrylic resins and such as polyacrylamide and latex beads. Techniquesfor coupling antibodies to such solid supports are well known in the art(Weir et al., “Handbook of Experimental Immunology” 4th Ed., BlackwellScientific Publications, Oxford, England, Chapter 10, 1986; Jacoby etal., Meth. Enzym. 34, Academic Press, N.Y., 1974). The immobilizedantibodies of the present invention can be used for in vitro, in vivo,and in situ assays as well as in immunochromotography.

[0144] Furthermore, one skilled in the art can readily adapt currentlyavailable procedures, as well as the techniques, methods and kitsdisclosed above with regard to antibodies, to generate peptides capableof binding to a specific peptide sequence in order to generaterationally designed antipeptide peptides, for example see Hurby et al.,“Application of Synthetic Peptides: Antisense Peptides”, In SyntheticPeptides, A User's Guide, W. H. Freeman, NY, pp. 289-307(1992), andKaspczak et al., Biochemistry 28:9230-8(1989).

[0145] VII. An Antibody Based Method and Kit for Detecting ALP

[0146] The present invention encompasses a method of detecting an ALPpolypeptide in a sample, comprising incubating a test sample with one ormore of the antibodies of the present invention and determining whetherthe antibody binds to the test sample. The method can include the stepsof, for example: (a) contacting the sample with an above-describedantibody, under conditions such that immunocomplexes form, and (b)detecting the presence of said antibody bound to the polypeptide.Altered levels, either an increase or decrease, of ALP in a sample ascompared to normal levels may indicate disease.

[0147] Conditions for incubating an antibody with a test sample vary.Incubation conditions depend on the format employed in the assay, thedetection methods employed, and the type and nature of the antibody usedin the assay. One skilled in the art will recognize that any one of thecommonly available immunological assay formats (such asradioimmunoassays, enzyme-linked immunosorbent assays, diffusion basedOuchterlony, or rocket immunofluorescent assays) can readily be adaptedto employ the antibodies of the present invention. Examples of suchassays can be found in Chard, “An Introduction to Radioimmunoassay andRelated Techniques” Elsevier Science Publishers, Amsterdam, TheNetherlands (1986); Bullock et al., “Techniques in Immunocytochemistry,”Academic Press, Orlando, Fla. Vol. 1(1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, “Practice and Theory of Enzyme Immunoassays: LaboratoryTechniques in Biochemistry and Molecular Biology,” Elsevier SciencePublishers, Amsterdam, The Netherlands (1985).

[0148] The immunological assay test samples of the present inventioninclude cells, protein or membrane extracts of cells, or biologicalfluids such as blood, serum, plasma, or urine. The test sample used inthe above-described method will vary based on the assay format, natureof the detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing protein extracts or membraneextracts of cells are well known in the art and can be readily adaptedin order to obtain a sample which is compatable with the systemutilized.

[0149] A kit contains all the necessary reagents to carry out thepreviously described methods of detection. The kit may comprise: (i) afirst container containing an above-described antibody, and (ii) secondcontainer containing a conjugate comprising a binding partner of theantibody and a label. In another preferred embodiment, the kit furthercomprises one or more other containers comprising one or more of thefollowing: wash reagents and reagents capable of detecting the presenceof bound antibodies.

[0150] Examples of detection reagents include, but are not limited to,labeled secondary antibodies, or in the alternative, if the primaryantibody is labeled, the chromophoric, enzymatic, or antibody bindingreagents which are capable of reacting with the labeled antibody. Thecompartmentalized kit may be as described above for nucleic acid probekits. One skilled in the art will recognize that the antibodiesdescribed in the present invention can readily be incorporated into oneof the established kit formats which are well known in the art.

[0151] VIII. Isolation of Natural Binding Partners of ALP

[0152] The present invention also relates to methods of detectingnatural binding partners capable of binding to an ALP polypeptide. Anatural binding partner of ALP may be, for example, an SH3 domaincontaining protein which can interact with an ALP proline-rich domain aspart of a signaling cascade. The binding parter(s) may be present withina complex mixture, for example, serum, body fluids, or cell extracts.

[0153] In general methods for identifying natural binding partnerscomprise incubating a substance with ALP and detecting the presence of asubstance bound to ALP. Preferred methods include the two-hybrid systemof Fields and Song (supra) and co-immunoprecipitation wherein an ALPpolypeptide is allowed to bind to a natural binding partner, then thepolypeptide complex is immunoprecipitated using ALP-specific antibodies.The natural binding partner can then be isolated and identified bytechniques well known in the art.

[0154] IX. Identification of and Uses for Substances Capable ofModulating ALP Activity

[0155] The present invention also relates to a method of detecting asubstance capable of modulating ALP activity. Such substances can eitherenhance activity (agonists) or inhibit activity (antagonists). Agonistsand antagonists can be peptides, antibodies, products from naturalsources such as fungal or plant extracts or small molecular weightorganic compounds. In general, small molecular weight organic compoundsare preferred. Examples of classes of compounds that can be tested forALP modulating activity are, for example but not limited to, thiazoles(see for example co-pending U.S. application No. 60/033,522, filed Dec.19, 1996; No. 08/660,900, filed Jun. 7, 1996), and naphthopyrones (U.S.Pat. No. 5,602,171, issued Feb. 11, 1997).

[0156] In general the method comprises incubating cells that produce ALPin the presence of a test substance and detecting changes in the levelof ALP activity or ALP binding partner activity. A change in activitymay be manifested by increased or decreased phosphorylation of an ALPpolypeptide, increased or decreased phosphorylation of an ALP substrate,increased or decreased binding to an ALP natural binding partner orincreased or decreased biological response in cells. A method fordetecting modulation of ALP activity using the phosphorylation of anartificial substrate is shown in the examples below. Biologicalresponses can include, for example, proliferation, differentiation,survival, or motility. The substance thus identified would produce achange in activity indicative of the agonist or antagonist nature of thesubstance. Once the substance is identified it can be isolated usingtechniques well known in the art, if not already available in a purifiedform.

[0157] The present invention also encompasses a method of agonizing(stimulating) or antagonizing ALP associated activity in a mammalcomprising administering to said mammal an agonist or antagonist to ALPin an amount sufficient to effect said agonism or antagonism. Alsoencompassed in the present application is a method of treating diseasesin a mammal with an agonist or antagonist of ALP-related activitycomprising administering the agonist or antagonist to a mammal in anamount sufficient to agonize or antagonize ALP associated function(s).The particular compound can be administered to a patient either byitself or in a pharmaceutical composition where it is mixed withsuitable carriers or excipient(s). In treating a patient, atherapeutically effective dose of the compound is administered. Atherapeutically effective dose referes to that amount of the compoundthat results in amelioration of symptioms or a prolongation of survivalin a patient.

[0158] Toxicity and therapeutic efficacy of such compounds can bedetermined by standard pharmaceutical procedures in cell cultures crexperimental animals. Cell clultur3 assays and animal studies can beused for determining the LD₅₀ (the dose lethal to 50% of a population)and the ED₅₀ (the dose therapeutically effective in 50% of apopulation). The dose ratio between toxic and therapeutic effects is thetherapeutic index, which can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit large therapeutic indices are preferred. Thedata obtained from these cell culture assays and animal studies can beused in formulating a range of dosages for use in human. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

[0159] For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays by determining an IC₅₀ (i.e., the concentration of thetest compound which achieves a half-maximal disruption of the proteincomplex, or a half-maximal inhibition of the cellular level and/oractivity of a cellular component, ex. ALP). A dose can then beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ as determined in cellculture. Such information can be used to more accurately determineuseful doses in humans. Levels in plasma may be measured, for example,by HPLC. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g. Fingl et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1, p. 1).

[0160] It should be noted that the attending physician would know how toand when to terminate, interrupt, or adjust administration due totoxicity, or to organ dysfunctions. Conversely, the attending physicianwould also know to adjust treatment to higher levels if the clinicalresponse were not adequate (precluding toxicity). The magnitude of anadministrated dose in the management of the oncogenic disorder ofinterest will vary with the severity of the condition to be treated andto the route of administration. The severity of the condition may, forexample, be evaluated, in part, by standard prognostic evaluationmethods. Further, the dose and perhaps dose frequency, will also varyaccording to the age, body weight, and response of the individualpatient. A program comparable to that discussed above may be used inveterinary medicine.

[0161] Depending on the specific conditions being treated, such agentsmay be formulated and administered systemically or locally. Techniquesfor formulation and administration may be found in “Remington'sPharmaceutical Sciences,” 1990, 18th ed., Mack Publishing Co., Easton,Pa. Suitable routes may include oral, rectal, transdermal, vaginal,transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intramedullary injections, aswell as intrathecal, direct intraventricular, intravenous,intraperitoneal, intranasal, or intraocular injections, just to name afew.

[0162] For injection, the agents of the invention may be formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hanks's solution, Ringer's solution, or physiological saline buffer.For such transmucosal administration, penetrants appropriate to thebarrier to be permeated are used in the formulation. Such penetrants aregenerally known in the art.

[0163] Use of pharmaceutically acceptable carriers to formulate thecompounds herein disclosed for the practice of the invention intodosages suitable for systemic administration is within the scope of theinvention. With proper choice of carrier and suitable manufacturingpractice, the compositions of the present invention, in particular thoseformulated as solutions, may be administered parenterally, such as byintravenous injection. The compounds can be formulated readily usingpharmaceutically acceptable carriers well known in the art into dosagessuitable for oral administration. Such carriers enable the compounds ofthe invention to be formulated as tablets, pills, capsules, liquids,gels, syrups, slurries, suspensions and the like, for oral ingestion bya patient to be treated. Particlar formulations suitable for parenteraladministration of hydrophobic compounds can be found in U.S. Pat. No.5,610,173, issued Mar. 11, 1997 and U.S. Provisional Application SerialNo. 60/039,870, filed Mar. 5, 1997, both of which are incorporated byreference herein in their entirety.

[0164] Agents intended to be administered intracellularly may beadministered using techniques well known to those of ordinary skill inthe art. For example, such agents may be encapsulated into liposomes,then administered as described above. Liposomes are spherical lipidbilayers with aqueous interiors. All molecules present in an aqueoussolution at the time of liposome formation are incorporated into theaqueous interior. The liposomal contents are both protected from theexternal microenvironment and, because liposomes fuse with cellmembranes, are efficiently delivered into the cell cytoplasm. Smallorganic molecules may be directly administered intracellularly due totheir hydrophobicity.

[0165] Pharmaceutical compositions suitable for use in the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve its intended purpose.Determination of an effective amount is well within the capability ofthose skilled in the art, especially in light of the detailed disclosureprovided herein.

[0166] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.The preparations formulated for oral administration may be in the formof tablets, dragees, capsules, or solutions.

[0167] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.

[0168] Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

[0169] Pharmaceutical preparations for oral use can be obtained bycombining the active compounds with solid excipient, optionally grindinga resulting mixture, and processing the mixture of granules, afteradding suitable auxiliaries, if desired, to obtain tablets or drageecores. Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol; cellulosepreparations such as, for example, maize starch, wheat starch, ricestarch, potato starch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate.

[0170] Dragee cores are provided with suitable coatings. For thispurpose, concentrated sugar solutions may be used, which may optionallycontain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel,polyethylene glycol, and/or titanium dioxide, lacquer solutions, andsuitable organic solvents or solvent mixtures. Dyestuffs or pigments maybe added to the tablets or dragee coatings for identification or tocharacterize different combinations of active compound doses.

[0171] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added.

[0172] X. Transgenic Animals

[0173] Also contemplated by the invention are transgenic animals usefulfor the study of ALP activity in complex in vivo systems. A “transgenicanimal” is an animal having cells that contain DNA which has beenartificially inserted into a cell, which DNA becomes part of the genomeof the animal which develops from that cell. Preferred transgenicanimals are primates, mice, rats, cows, pigs, horses, goats, sheep, dogsand cats. The transgenic DNA may encode for a human ALP polypeptide.Native expression in an animal may alternatively be reduced by providingan amount of antisense RNA or DNA effective to reduce expression of thetarget gene.

[0174] A variety of methods are available for the production oftransgenic animals associated with this invention. DNA sequencesencoding ALP can be injected into the pronucleus of a fertilized eggbefore fusion of the male and female pronuclei, or injected into thenucleus of an embryonic cell (e.g., the nucleus of a two cell embryo)following the initiation of cell division (Brinster, et al., Proc. Nat.Acad. Sci. USA 82: 4438, 1985). Embryos can be infected with viruses,especially retroviruses, modified to carry inorganic-ion receptornucleotide sequences of the invention.

[0175] Pluripotent stem cells derived from the inner cell mass of theembryo and stabilized in culture can be manipulated in culture toincorporate nucleotide sequences of the invention. A transgenic animalcan be produced from such cells through implantation into a blastocystthat is implanted into a foster mother and allowed to come to term.Animals suitable for transgenic experiments can be obtained fromstandard commercial sources such as Charles River (Wilmington, Mass.),Taconic (Germantown, N.Y.), Harlan Sprague Dawley (Indianapolis, Ind.),etc.

[0176] The procedures for manipulation of the rodent embryo and formicroinjection of DNA into the pronucleus of the zygote are well knownto those of ordinary skill in the art (Hogan, et al., supra).Microinjection procedures for fish, amphibian eggs and birds aredetailed in Houdebine and Chourrout, Experientia 47: 897-905, 1991).Other procedures for introduction of DNA into tissues of animals aredescribed in U.S. Pat. No., 4,945,050 (Sandford et al., Jul. 30, 1990).

[0177] By way of example only, to prepare a transgenic mouse, femalemice are induced to superovulate. After being allowed to mate, thefemales are sacrificed by CO₂ asphyxiation or cervical dislocation andembryos are recovered from excised oviducts. Surrounding cumulus cellsare removed. Pronuclear embryos are then washed and stored until thetime of injection. Randomly cycling adult female mice are paired withvasectomized males. Recipient females are mated at the same time asdonor females. Embryos then are transferred surgically. The procedurefor generating transgenic rats is similar to that of mice. See Hammer,et al., Cell 63:1099-1112, 1990).

[0178] Methods for the culturing of embryonic stem (ES) cells and thesubsequent production of transgenic animals by the introduction of DNAinto ES cells using methods such as electroporation, calciumphosphate/DNA precipitation and direct injection also are well known tothose of ordinary skill in the art. (See, for example, Teratocarcinomasand Embryonic Stem Cells, A Practical Approach, E. J. Robertson, ed.,IRL Press, 1987). In cases involving random gene integration, a clonecontaining the sequence(s) of the invention is co-transfected with agene encoding resistance. Alternatively, the gene encoding neomycinresistance is physically linked to the sequence(s) of the invention.Transfection and isolation of desired clones are carried out by any oneof several methods well known to those of ordinary skill in the art (E.J. Robertson, supra). DNA molecules introduced into ES cells can also beintegrated into the chromosome through the process of homologousrecombination. (Capecchi, Science 244:1288, 1989). Methods for positiveselection of the recombination event (i.e., neo resistance) and dualpositive-negative selection (i.e., neo resistance and gancyclovirresistance) and the subsequent identification of the desired clones byPCR have been described by Capecchi, supra and Joyner et al., (Nature338:153, 1989), the teachings of which are incorporated by referenceherein. The final phase of the procedure is to inject targeted ES cellsinto blastocysts and to transfer the blastocysts into pseudopregnantfemales. The resulting chimeric animals are bred and the offspring areanalyzed by Southern blotting to identify individuals that carry thetransgene. Procedures for the production of non-rodent mammals and otheranimals have been discussed by others. (See Houdebine and Chourrout,supra; Pursel, et al., Science 244:1281, 1989; Simms, et al.,Bio/Technology 6:179, 1988).

[0179] Thus, the invention provides transgenic, nonhuman mammalscontaining a transgene encoding an ALP polypeptide or a gene effectingthe expression of an ALP polypeptide. Such transgenic nonhuman mammalsare particularly useful as an in vivo test system for studying theeffects of introducing an ALP polypeptide, regulating the expression ofan ALP polypeptide (i.e., through the introduction of additional genes,antisense nucleic acids, or ribozymes).

[0180] XI. Gene Therapy

[0181] ALP nucleic acid sequences, both mutated and non-mutated, willalso be useful in gene therapy (reviewed in Miller, Nature 357:455-460,(1992). Miller states that advances have resulted in practicalapproaches to human gene therapy that have demonstrated positive initialresults. The basic science of gene therapy is described in Mulligan,Science 260:926, 1993. As used herein “gene therapy” is a form of genetransfer and is included within the definition of gene transfer as usedherein and specifically refers to gene transfer to express a therapeuticproduct from a cell in vivo or in vitro. Gene transfer can be performedex vivo on cells which are then transplanted into a patient, or can beperformed by direct administration of the nucleic acid or nucleicacid-protein complex into the patient.

[0182] In one preferred embodiment, an expression vector containing anALP coding sequence or an ALP mutant coding sequence as described aboveis inserted into cells, the cells are grown in vitro and then infused inlarge numbers into patients. In another preferred embodiment, a DNAsegment containing a promoter of choice (for example a strong promoter)is transferred into cells containing an endogenous ALP in such a mannerthat the promoter segment enhances expression of the endogenous ALP gene(for example, the promoter segment is transferred to the cell such thatit becomes directly linked to the endogenous ALP gene).

[0183] The gene therapy may involve the use of an adenovirus containingALP cDNA targeted to an appropriate cell type, systemic ALP increase byimplantation of engineered cells, injection with ALP virus, or injectionof naked ALP DNA into appropriate cells or tissues, for example neurons.

[0184] Expression vectors derived from viruses such as retroviruses,vaccinia virus, adenovirus, adeno-associated virus, herpes viruses,other RNA viruses, or bovine papilloma virus, may be used for deliveryof nucleotide sequences (e.g., cDNA) encoding recombinant ALP proteininto the targeted cell population (e.g., tumor cells or neurons).Methods which are well known to those skilled in the art can be used toconstruct recombinant viral vectors containing coding sequences. See,for example, the techniques described in Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y.(1989), and in Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates and Wiley Interscience, N.Y. (1989).Alternatively, recombinant nucleic acid molecules encoding proteinsequences can be used as naked DNA or in reconstituted system, e.g.,liposomes or other lipid systems for delivery to target cells (See e.g.,Felgner et al., Nature 337:387-8, 1989). Several other methods for thedirect transfer of plasmid DNA into cells exist for use in human genetherapy and involve targeting the DNA to receptors on cells bycomplexing the plasmid DNA to proteins. See, Miller, supra.

[0185] In its simplest form, gene transfer can be performed by simplyinjecting minute amounts of DNA into the nucleus of a cell, through aprocess of microinjection. (Capecchi MR, Cell 22:479-88, 1980). Oncerecombinant genes are introduced into a cell, they can be recognized bythe cells normal mechanisms for transcription and translation, and agene product will be expressed. Other methods have also been attemptedfor introducing DNA into larger numbers of cells. These methods include:transfection, wherein DNA is precipitated with CaPO₄ and taken intocells by pinocytosis (Chen C. and Okayama H, Mol. Cell Biol. 7:2745-52,1987); electroporation, wherein cells are exposed to large voltagepulses to introduce holes into the membrane (Chu G., et al., NucleicAcids Res., 15:1311-26, 1987); lipofection/liposome fusion, wherein DNAis packaged into lipophilic vesicles which fuse with a target cell(Felgner P L., et al., Proc. Natl. Acad. Sci. USA. 84:7413-7, 1987));and particle bombardment using DNA bound to small projectiles (Yang N S.et al., Proc. Natl. Acad. Sci. 87:9568-72, 1990). Another method forintroducing DNA into cells is to couple the DNA to chemically modifiedproteins.

[0186] It has also been shown that adenovirus proteins are capable ofdestabilizing endosomes and enhancing the uptake of DNA into cells. Theadmixture of adenovirus to solutions containing DNA complexes, or thebinding of DNA to polylysine covalently attached to adenovirus usingprotein crosslinking agents substantially improves the uptake andexpression of the recombinant gene. Curiel D T.

[0187] As used herein “gene transfer” means the process of introducing aforeign nucleic acid molecule into a cell. Gene transfer is commonlyperformed to enable the expression of a particular product encoded bythe gene. The product may include a protein, polypeptide, antisense DNAor RNA, or enzymatically active RNA. Gene transfer can be performed incultured cells or by direct administration into animals. Generally genetransfer involves the process of nucleic acid contact with a target cellby non-specific or receptor mediated interactions, uptake of nucleicacid into the cell through the membrane or by endocytosis, and releaseof nucleic acid into the cytoplasm from the plasma membrane or endosome.Expression may require, in addition, movement of the nucleic acid intothe nucleus of the cell and binding to appropriate nuclear factors fortranscription.

[0188] In another preferred embodiment, a vector having nucleic acidsequences encoding an ALP is provided in which the nucleic acid sequenceis expressed only in specific tissue. Methods of achievingtissue-specific gene expression as set forth in InternationalPublication No. WO 93/09236, filed Nov. 3, 1992 and published May 13,1993.

[0189] In all of the preceding vectors set forth above, a further aspectof the invention is that the nucleic acid sequence contained in thevector may include additions, deletions or modifications to some or allof the sequence of the nucleic acid, as defined above.

[0190] In another preferred embodiment, a method of gene replacement isset forth. “Gene replacement” as used herein means supplying a nucleicacid sequence which is capable of being expressed in vivo in an animaland thereby providing or augmenting the function of an endogenous genewhich is missing or defective in the animal.

[0191] XII. Compounds that Modulate the Function of ALP Proteins

[0192] In an effort to discover novel treatments for diseases,biomedical researchers and chemists have designed, synthesized, andtested molecules that inhibit the function of protein kinases. Somesmall organic molecules form a class of compounds that modulate thefunction of protein kinases. Examples of molecules that have beenreported to inhibit the function of protein kinases include, but are notlimited to, bis monocyclic, bicyclic or heterocyclic aryl compounds (PCTWO 92/20642, published Nov. 26, 1992 by Maguire et al.),vinylene-azaindole derivatives (PCT WO 94/14808, published Jul. 7, 1994by Ballinari et al.), 1-cyclopropyl-4-pyridyl-quinolones (U.S. Pat. No.5,330,992), styryl compounds (U.S. Pat. No. 5,217,999),styryl-substituted pyridyl compounds (U.S. Pat. No. 5,302,606), certainquinazoline derivatives (EP Application No. 0 566 266 A1), seleoindolesand selenides (PCT WO 94/03427, published Feb. 17, 1994 by Denny atal.), tricyclic polyhydroxylic compounds (PCT WO 92/21660, publishedDec. 10, 1992 by Dow), and benzylphosphonic acid compounds (PCT WO91/15495, published Oct. 17, 1991 by Dow et al). The compounds that cantraverse cell membranes and are resistant to acid hydrolysis arepotentially advantageous therapeutics as they can become highlybioavailable after being administered orally to patients. However, manyof these protein kinase inhibitors only weakly inhibit the function ofprotein kinases. In addition, many inhibit a variety of protein kinasesand will therefore cause multiple side-effects as therapeutics fordiseases.

[0193] Some indolinone compounds, however, form classes of acidresistant and membrane permeable organic molecules. WO 96/22976,published Aug. 1, 1996 by Ballinari et al. describes hydrosolubleindolinone compounds that harbor tetralin, naphthalene, quinoline, andindole substituents fused to the oxindole ring. These bicyclicsubstituents are in turn substituted with polar moieties includinghydroxylated alkyl, phosphate, and ether moieties. U.S. patentapplication Ser. No. 08/702,232, filed Aug. 23, 1996, entitled“Indolinone Combinatorial Libraries and Related Products and Methods forthe Treatment of Disease” by Tang et al. (Lyon & Lyon Docket No.221/187) and Ser. No. 08/485,323, filed Jun. 7, 1995, entitled“Benzylidene-Z-Indoline Compounds for the Treatment of Disease” by Tanget al. (Lyon & Lyon Docket No. 223/298) and International PatentPublication WO 96/22976, published Aug. 1, 1996 by Ballinari et al., allof which are incorporated herein by reference in their entirety,including any drawings, describe indolinone chemical libraries ofindolinone compounds harboring other bicyclic moieties as well asmonocyclic moieties fused to the oxindole ring. application Ser. No.08/702,232, filed Aug. 23, 1996, entitled “Indolinone CombinatorialLibraries and Related Products and Methods for the Treatment of Disease”by Tang et al. (Lyon & Lyon Docket No. 221/187), Ser. No. 08/485,323,filed Jun. 7, 1995, entitled “Benzylidene-Z-Indoline Compounds for theTreatment of Disease” by Tang et al. (Lyon & Lyon Docket No. 223/298),and WO 96/22976, published Aug. 1, 1996 by Ballinari et al. teachmethods of indolinone synthesis, methods of testing the biologicalactivity of indolinone compounds in cells, and inhibition patterns ofindolinone derivatives.

[0194] Other examples of substances capable of modulating ALP activityinclude, but are not limited to, tyrphostins, quinazolines,quinoxolines, and quinolines.

[0195] The quinazolines, tyrphostins, quinolines, and quinoxolinesreferred to above include well known compounds such as those describedin the literature. For example, representative publications describingquinazoline include Barker et al., EPO Publication No. 0 520 722 A1;Jones et al., U.S. Pat. No. 4,447,608; Kabbe et al., U.S. Pat. No.4,757,072; Kaul and Vougioukas, U.S. Pat. No. 5, 316,553; Kreighbaum andComer, U.S. Pat. No. 4,343,940; Pegg and Wardleworth, EPO PublicationNo. 0 562 734 A1; Barker et al., Proc. of Am. Assoc. for Cancer Research32:327 (1991); Bertino, J. R., Cancer Research 3:293-304 (1979);Bertino, J. R., Cancer Research 9(2 part 1):293-304 (1979); Curtin etal., Br. J. Cancer 53:361-368 (1986); Fernandes et al., Cancer Research43:1117-1123 (1983); Ferris et al. J. Org. Chem. 44(2):173-178; Fry etal., Science 265:1093-1095 (1994); Jackman et al., Cancer Research51:5579-5586 (1981); Jones et al. J. Med. Chem. 29(6):1114-1118; Lee andSkibo, Biochemistry 26 (23):7355-7362 (1987); Lemus et al., J. Org.Chem. 54:3511-3518 (1989); Ley and Seng, Synthesis 1975:415-522 (1975);Maxwell et al., Magnetic Resonance in Medicine 17:189-196 (1991); Miniet al., Cancer Research 45:325-330 (1985); Phillips and Castle, J.Heterocyclic Chem. 17(19):1489-1596 (1980); Reece et al., CancerResearch 47(11):2996-2999 (1977); Sculier et al., Cancer Immunol. andImmunother. 23:A65 (1986); Sikora et al., Cancer Letters 23:289-295(1984); Sikora et al., Analytical Biochem. 172:344-355 (1988); all ofwhich are incorporated herein by reference in their entirety, includingany drawings.

[0196] Quinoxaline is described in Kaul and Vougioukas, U.S. Pat. No.5,316,553, incorporated herein by reference in its entirety, includingany drawings.

[0197] Quinolines are described in Dolle et al., J. Med. Chem.37:2627-2629 (1994); MaGuire, J. Med. Chem. 37:2129-2131 (1994); Burkeet al., J. Med. Chem. 36:425-432 (1993); and Burke et al. BioOrganicMed. Chem. Letters 2:1771-1774 (1992), all of which are incorporated byreference in their entirety, including any drawings.

[0198] Tyrphostins are described in Allen et al., Clin. Exp. Immunol.91:141-156 (1993); Anafi et al., Blood 82:12:3524-3529 (1993); Baker etal., J. Cell Sci. 102:543-555 (1992); Bilder et al., Amer. Physiol. Soc.pp. 6363-6143:C721-C730 (1991); Brunton et al., Proceedings of Amer.Assoc. Cancer Rsch. 33:558 (1992); Bryckaert et al., Experimental CellResearch 199:255-261 (1992); Dong et al., J. Leukocyte Biology 53:53-60(1993); Dong et al., J. Immunol. 151(5):2717-2724 (1993); Gazit et al.,J. Med. Chem. 32:2344-2352 (1989); Gazit et al., “J. Med. Chem.36:3556-3564 (1993); Kaur et al., Anti-Cancer Drugs 5:213-222 (1994);Kaur et al., King et al., Biochem. J. 275:413-418 (1991); Kuo et al.,Cancer Letters 74:197-202 (1993); Levitzki, A., The FASEB J. 6:3275-3282(1992); Lyall et al., J. Biol. Chem. 264:14503-14509 (1989); Peterson etal., The Prostate 22:335-345 (1993); Pillemer et al., Int. J. Cancer50:80-85 (1992); Posner et al., Molecular Pharmacology 45:673-683(1993); Rendu et al., Biol. Pharmacology 44(5):881-888 (1992); Sauro andThomas, Life Sciences 53:371-376 (1993); Sauro and Thomas, J. Pharm. andExperimental Therapeutics 267(3):119-1125 (1993); Wolbring et al., J.Biol. Chem. 269(36):22470-22472 (1994); and Yoneda et al., CancerResearch 51:4430 4435 (1991); all of which are incorporated herein byreference in their entirety, including any drawings.

EXAMPLES EXAMPLE 1 Isolation of cDNA Clones Encoding ALP

[0199] The example below describes the isolation and identification of anew PTP sequence from mouse tissues and the subsequent cloning of afull-length human ALP. Also described are probes useful for thedetection of ALP in cells or tissues.

[0200] Materials and Methods:

[0201] Total RNAs were isolated using a commonly known guanidinesalts/phenol extraction protocol from normal mouse fat and ratpituitary. Chomczynski & Sacchi, 1987, Anal. Biochem. 162: 156. TheseRNA extracts were used to generate single-stranded cDNA using theSuperscript Pre-amplification System (GIBCO BRL, Gaithersburg, Md.;Gerard et al., 1989, FOCUS 11: 66) under conditions recommended by themanufacturer. a typical reaction used 10 μg total RNA with 1.5 μgoligo(dT)₁₂₋₁₈ in a reaction volume of 60 μL. The product was treatedwith RNaseH and diluted to 100 μL with H₂O. For subsequent PCRamplification, 1-4 μL of this sscDNA was used in each reaction.

[0202] Degenerate oligonucleotides were synthesized on an AppliedBiosystems 394 DNA synthesizer using established phosphoramiditechemistry, precipitated with ethanol and used unpurified for PCR. Thesequence of the degenerate oligonucleotide primers were as follows:

[0203] PTPDFW=5′-GAYTTYTGGVRNATGRTNTGGGA-3′ (SEQ ID NO:3)

[0204] PTPHCSAG=5′-CGGCCSAYNCCNGCNSWRCARTG-3′ (SEQ ID NO:4)

[0205] PTPYINA=5′-ATCCCCGGCTCTGAYTAYATHMAYGC-3′ (SEQ ID NO:5)

[0206] These primers were derived from the peptide sequencesDFWXMXW(E/D) (SEQ ID NO:6) (sense strand from PTP catalytic region) andHCXAGXG (SEQ ID NO:7) (antisense strand from PTP catalytic region), andIPGSDYI(N/H)A (SEQ ID NO:8) respectively. The standard UIPACdesignations for degenerate residue designations are: N=A, C, G, or T;R=A or G; Y=C or T; V=A, C or G; W=C or T; S=C or G; M=A or C; and H=A,C or T.

[0207] PCR reactions were performed using degenerate primers applied tothe single-stranded cDNA listed above. The primers were added at a finalconcentration of 5 μM each to a mixture containing 10 mM TrisHCl (pH8.3), 50 mM KCl, 1.5 mM MgCl₂, 200 mM each deoxynucleoside triphosphate,0.001% gelatin, 1.5 U AmpliTaq DNA Polymerase (Perkin-Elmer/Cetus), and1-4 μL cDNA. Following 3 min denaturation at 95° C., the cyclingconditions were 94° C. for 30 s, 50° C. for 1 min, and 72° C. for 1 min45 s for 35 cycles. PCR fragments migrating between 350-400 bp wereisolated from 2% agarose gels using the GeneClean Kit (Bio101), and T-Acloned into the PCRII vector (Invitrogen Corp. U.S.A.) according to themanufacturer's protocol.

[0208] Colonies were selected for mini plasmid DNA-preparations usingQiagen columns and the plasmid DNA was sequenced using cycle sequencingdye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City,Calif.). Sequencing reaction products were run on an ABI Prism 377 DNASequencer, and analyzed using the BLAST alignment algorithm. Altschul etal., J. Mol. Biol. 215: 403-410. A single clone encoding a novel PTP(S50-151), designated murine ALP, was isolated from murine adiposetissue using degenerate oligonucleotides PTPDFW (SEQ ID NO:3) andPTPHCSAG (SEQ ID NO:4), and a related rat ALP clone was isolated fromrat pituitary using degenerate oligonucleotides PTPYINA (SEQ ID NO:5)and PTPHCSAG (SEQ ID NO:4).

[0209] To isolate a full-length human ALP a human CDNA library wasconstructed in lambda ZapII (Stratagene, La Jolla, Calif.) frompolyA+RNA isolated from the human neuroblastoma cell line IMR32. Thelibrary was screened to isolate full-length transcripts encoding ALP.The murine ALP fragment was ³²P-labeled by random priming and used as ahybridization probe at 2×10⁶ cpm/mL following standard techniques forlibrary screening. Pre-hybridization (3 h) and hybridization (overnight)were conducted at 42° C. in 5× SSC, 5× Denhart's solution, 2.5% dextransulfate, 50 mM Na₂PO₄/NaHPO₄ [pH 7.0], 50% formamide with 100 mg/mLdenatured salmon sperm DNA. Stringent washes were performed at 65° C. in0.1× SSC with 0.1% SDS. Multiple clones were isolated and one 4.5 kbclone spanned the entire coding region of ALP. The final sequence wasverified by sequencing of both strands using a cycle sequencingdye-terminator kit with AmpliTaq DNA Polymerase, FS (ABI, Foster City,Calif.). Sequencing reaction products were run on an ABI Prism 377 DNASequencer.

[0210] Results:

[0211] The 4,456 bp human ALP nucleotide sequence encodes a polypeptideof 1,274 amino acids. The amino acid sequence shows no signal sequenceor a transmembrane domain and is therefore an intracellular protein. TheN-terminal end extends from amino acids 1-857 and contains severalputative tyrosine phosphorylation sites and a proline-rich region (30.6%prolines) from amino acids 353-777. This proline-rich region isdistantly related to plant extensin proteins (30.2% amino acid identitywith Zea mays extensin-like protein GB:Z34465 using Smith-Watermanalignment) and may represent a protein interaction domain as well as thesite for interaction with proteins containg SH3 motifs. The C-terminaltail of ALP extends from amino acid 1097-1274 and contains aproline/serine rich region (45.6% serines plus prolines from amino acids1101-1214) resembling a PEST motif. This region also could serve as atarget for binding proteins via their SH3 motifs.

[0212] The catalytic domain extends from amino acids 858-1096 and shares32-37% amino acid identity to PTPs from multiple subfamilies: TC-PTP(P17706: 37.1%) PTP-BAS (D21209: 32.9%), PTPα (M34668: 34.2%), PTPβ(P23467: 34.2%), PTPσ (A49104: 33.2%), PTP1B (P20417: 34.9%), suggestingthat it represents a new family of PTPs. While all other cytoplasmicPTPs have their catalytic domain at either the N- or C-terminal portionof the protein, ALP has a central catalytic domain flanked by large N-and C-terminal domains. Its catalytic domain conserves most of theinvariant residues present in other PTPs, but does has several atypicalamino acids. In ALP, the amino acid sequence HCSAG (SEQ ID NO:6), ischanged to HCSSG (amino acid positions 1029-1033) (SEQ ID NO:7). Thismotif is in the catalytic site of the crystal stucture of PTP1B andPTPa, and the Ala to Ser change may effect catalyitic activity orspecificty. ALP also has a change from WPD to WPE (amino acids positions993-995) in its predicted surface loop of the catalytic domain. In PTP1Bthis Aspartate participates in a salt bridge and falls into thecatalytic site on binding to a specific peptide substrate. This Asp toGlu alteration is also present in three other mammalian PTPs (PTPD1,PCP2, PTPS31).

EXAMPLE 2 Expression of ALP

[0213] The example below shows the evaluation of ALP expression innormal human tissues and in a wide variety of cancers.

[0214] Materials and Methods:

[0215] Northern blots were prepared by running 20 μg total RNA per laneisolated from 60 different tumor cell lines (HOP-92, EKVX, NCI-H23,NCI-H226, NCI-H322M, NCI-H460, NCI-H522, A549, HOP-62, OVCAR-3, OVCAR-4,OVCAR-5, OVCAR-8, IGROV1, SK-OV-3, SNB-19, SNB-75, U251, SF-268, SF-295,SF-539, CCRF-CEM, K-562, MOLT-4, HL-60, RPMI 8226, SR, DU-145, PC-3,HT-29, HCC-2998, HCT-116, SW620, Colo 205, HTC15, KM-12, UO-31, SN12C,A498, CaKil, RXF-393, ACHN, 786-0, TK-10, LOX IMVI, Malme-3M, SK-MEL-2,SK-MEL-5, SK-MEL-28, UACC-62, UACC-257, M14, MCF-7, MCF-7/ADR RES,Hs578T, MDA-MB-231, MDA-MB-435, MDA-N, BT-549, T47D). (obtained fromNick Scuidero, National Cancer Institute, Developmental TherapeuticsProgram, Rockville, Md.). The total RNA samples were run on a denaturingformaldehyde 1% agarose gel and transferred onto a nitrocellulosemembrane (BioRad, Calif.). Additional human normal tissue Northern blotscontaining 2 Ag polyA+ mRNA per lane from 16 different human normaltissues (thymus, lung, colon, testis, brain, heart, liver, pancreas,kidney, spleen, uterus, prostate, skeletal muscle, PBLs, placenta, smallintestine) on charge-modified nylon membranes (multiple tissue blots#7760-1 and #7766-1, Clontech, Palo Alto, Calif.) were also hybidized.

[0216] Nitrocellulose membranes for the total RNA samples werehybridized with randomly primed [gamma-³²P]dCTP-labeled probessynthesized from a 1 kb fragment of EcoRI-NotI of ALP. Hybridization wasperformed overnight at 42° C. in 4× SSPE, 2.5× Denhardt's solution, 50%formamide, 200 μg/mL denatured salmon sperm DNA, 100 μg/mL yeast tRNA(Boehringer Mannheim, Ind.), 0.2% SDS with 5×10⁶ cpm/mL of[gamma-³²P]dCTP-labeled DNA probe on a Techne Hybridizer H-1. The blotswere washed with 2× SSC, 0.1% SDS, at 65° C. for 20 min twice followedby 0.5× SSC in 0.1% SDS at 65° C. for 20 min. The blots were exposed toa phospho-imaging screen for 24 hours and scanned on a MolecularDynamics Phosphoimager SF.

[0217] For Clontech nylon-membrane blots, hybridization was performed at42° C. overnight in 5× SSC, 2% SDS, 10× Denhardt's solution, 50%formamide, 100 μg/mL denatured salmon sperm DNA with 1-2×10⁶ cpm/mL of[gamma-³²P]dCTP-labeled DNA probe. The blots were washed at roomtemperature in 2× SSC/0.05% SDS for 30 min and followed by at 50° C. in0.2× SSC/0.1% SDS for 30 min, and exposed for 48 hours on Kodak XAR-2film.

[0218] For analysis of expression using reverse-transcriptase-PCRdetection, total RNA was isolated from various cell lines or freshfrozen tissues by centrifugation through a cesium chloride cushion. 20μg of total RNA was reverse transcribed with random hexamers and Maloneyhuman leukemia virus reverse transcriptase (Super-ScriptII, GIBCO BRL,Gaithersburg, Md.). PCR was then used to amplify cDNA encoding ALP.Reverse transcriptase PCR (RT-PCR) reactions lacking only the reversetranscriptase were performed as controls. PCR products wereelectrophoresed on 3% agarose gels, visualized by ethidium bromidestaining and photographed on a UV light box.

[0219] The intensity of the fragment specific to ALP were compared amongdifferent RNA samples. A rating of 4 represents large quantities of ALPtranscript while a rating of 0 represents little or none of thetranscript was detected. It should be noted that detection of proteinsby RT-PCR indicates a relatively higher abundance than detection byNorthern blot as the RT-PCR technique utilizes total RNA whereasNorthern blot analysis is performed using an enriched RNA source (mRNA).

[0220] Results:

[0221] A single ALP mRNA transcript of approximately 5.0 kb wasvisualized by Northern analysis. This transcript was identified in mostof the normal tissue samples tested. However, the Northern analysisresults shown in the Table 1 illustrate that the relative abundance ofALP mRNA is quite divergent. In normal tissues, ALP was identified inhighest quantities in pancreas, followed by heart, testis, and skeletalmuscle. Lower levels of the ALP transcript were identified in placenta,thymus, lung, brain, liver, spleen, uterus, prostate and smallintestine. None of the ALP transcript was detected in colon, kidney andperipheral blood leucocytes (PBLs). ALP expression was also detected innormal human adipocytes by RT-PCR methods.

[0222] In Northern blots of total RNA from human tumor cell lines, theALP RNA transcript was most abundant in NCI-H226 (lung tumor), SK-OV-3(ovarian tumor), and RPMI 8226 (leukemia) cell lines. The transcript wasidentified at lower amounts in SNB-19 (CNS tumor), SF-268 (CNS tumor),SN12C (kidney tumor), SK-MEL-2 (melanoma), UACC-62 (melanoma), andUACC-257 (melanoma) cell lines. The ALP transcript was not detected inthe remaining of 44 human tumor cell lines. A summary of expression ofALP is shown in Table 1 below. TABLE 1 Cell type Origin ALP ThymusNormal tissue 0.5* Lung Normal tissue 0.5* Colon Normal tissue 0* TestisNormal tissue 2* Brain Normal tissue 0.5* Heart Normal tissue 2* LiverNormal tissue 0.5* Pancreas Normal tissue 3* Kidney Normal tissue 0*Spleen Normal tissue 0.5* Uterus Normal tissue 0.5* Prostate Normaltissue 0.5* Skeletal muscle Normal tissue 2* PBLs Normal tissue 0*Placenta Normal tissue 1* Small intestine Normal tissue 0.5* NCI-H226Lung tumor 4 SK-OV-3 Ovarian tumor 3 SNB-19 CNS tumor 2 U251 CNS tumor 1SF-268 CNS tumor 2 RPMI 8226 Leukemia 3 HTC15 Colon tumor 1 UO-31 Colontumor 1 SN12C Kidney tumor 2 SK-MEL-2 Melanoma 2 SK-MEL-28 Melanoma 1UACC-62 Melanoma 2 UACC-257 Melanoma 2 T47D Breast tumor 1

[0223] ALP exhihits increased expression in tumor cells compared totheir normal tissue counterparts. This differential expression suggestsa possible disregulation or involvement of ALP in development ormaintenance of the transformed phenotype.

EXAMPLE 3 Recombinant Expression of ALP

[0224] The following example illustrates the contruction of vectors forexpression of recombinant ALP and the creation of recombinant cell linesexpressing ALP.

[0225] Contruction of Expression Vectors

[0226] Expression constructs were generated by PCR-assisted mutagenesisin which the entire coding regions of ALP was introduced into themammalian expression vectors pcDNAIII (Invitrogen) for transientexpression analysis. Additional ALP constructs were made byoligonucleotide based PCR mutagenesis to convert atypical residues inthe PTP-related domain back to the amino acids more commonly present inother catalytically active PTPs. These changes include: His to Tyr atamino acid 861 (See SEQ ID NO:2); Ala to Gly at amino acid 902; Phe totrp at amino acid 941; Glu to Asp at amino acid 995; and Ser to Ala atamino acid 1032. Additional constructs containing paired mutations asabove were generated for amino acid positions 941/1032 and 902/1032.These constructs were ligated into the pcDNAIII mammalian expressionvector behind the CMV promoter.

[0227] The entire ALP open reading frame excluding the initiatingmethionines was generated by PCR and ligated into pGEX vector (PharmaciaBiotech, Upsala, Sweden) for bacterial production of GST-Lusion proteinsfor immunization of rabbits for antibody production. This vectorcontains the glutathione-S-transferase coding sequence followed by apolylinker for generating recombinant fusion proteins. The GST moietycomprises the N-terminal portion of the fusion protein. The various ALPmutants were also inseted into the pGEX vecotr for production ofrecombinant protein reagents.

[0228] Transient Expression in Mammalian Cells

[0229] The pcDNAIII expression plasmids (10 μg DNA/100 mm plate)containing the wild-type and mutant forms of the ALP gene wereintroduced into 293 cells with lipofectamine (Gibco BRL). After 72hours, the cells were harvested in 0.5 mL solubilization buffer (20 mMHEPES pH 7.35, 150 mM NaCl, 10% glycerol, l Triton X-100, 1.5 mM MgCl₂₁1 mM EGTA, 2 mM phenylmethylsulfonyl fluoride, 1 μg/mL aprotinin).Sample aliquots were resolved by SDS polyacrylamide gel electrophoresis(PAGE) on 15% acrylamide/0.5% bis-acrylamide gels andelectrophoretically transferred to nitrocellulose. Non-specific bindingwas blocked by preincubating blots in Blotto (phosphate buffered salinecontaining 5% w/v non-fat dried milk and 0.2% v/v nonidet P-40 (Sigma)),and recombinant protein was detected using antisera specific to theamino-terminal 352 residues (see below). Recombinant ALP proteinmigrated approximately 180 kDa, consistent with the predicted molecularweight of the 1274 amino acid protein.

[0230] Endogenous ALP was detected as a 200 kD protein in Western blotsof lysates from a variety of tumor cell lines including humanglioblastomas (U87MG, ATCC HTB 14; U118MG, ATCC HTB 15; U138MG, ATCC HTB16; A172, ATCC CRL 1620; Hs683, ATCC HTB 138), rodent gliomas (C6, ATCC107), rodent pituitary tumors (ATT20, ATCC CCL 89; GH3, ATCC CCL 82.1),human neuroblastomas (SKNMC, ATCC HTB 10; IMR 32, ATCC CCL 127), androdent adrenal pheochromocytomas (PC12, ATCC CRL 1721). ALP proteincould not be immunoprecipitated from the non-transformed cell line NIH3T3 (ATCC CRL 1658).

[0231] It is unclear why native ALP protein appears to be larger (200kDa) than recombinant ALP detected in transfected 293 cells (180 kDa).The difference could be the result of alternative RNA splicing, or apost-translational modification in the cell lines where it isendogenously expressed. Preliminary experiments indicate that ALP isphosphorylated on serine and threonine residues in transfected 293cells. In addition, several tyrosine-phosphorylated proteins areassociated with ALP since they are detected in Western blots using ananti-phosphotyrosine antibody following immunoprecipitation ofendogenous ALP from human tumor cell lines such as IMR32 aftertreatments with the phosphatase inhibitor pervanadate.

[0232] Generation of Virus Producing Cell Lines

[0233] pLXSN recombinant constructs containing the ALP gene aretransfected into an amphotropic helper cell line PA317 using CaCl₂mediated transfection. After selection on G418, the cells are plated onnormal media without G418 (500 μg/mL). Supernatants from resistant cellsare used to infect the ecotropic helper cell line GP+E86, and cellsagain selected on G418. Resistant cells are again taken off G418, andthe supernatants harvested every 8-12 hours and pooled as virus stock.Redemann et al., 1992, Mol. Cell. Biol. 12: 491-498. Viral stock titersare typically ˜106/mL.

[0234] Stable Expression in Mammalian Cells

[0235] NIH-3T3, BALB/3T3 or other suitable cells are grown in 100 mmplates with DMEM (Gibco) containing 10% fetal calf serum (FCS). Thecells are superinfected with the ALP retrovirus by adding approximately3 mL viral supernatant to 15 mL culture media for approximately 24hours. Cells expressing the retroviral constructs are then selected bygrowth in DMEM/10% FCS supplemented with 500 μg/mL G418.

EXAMPLE 4 Generation of Anti-ALP Antibodies

[0236] ALP-specific immunoreagents were generated by immunizing rabbitswith the bacterially expressed N-terminal 352 amino acid portion of ALPexpressed as a GST-fusion protein. Fusion protein was affinity purifiedusing glutathione-sepharose colums (Pharmacia). Polyclonal anti-serumagainst the N-terminal portion of ALP was generated by repeatedlyimmunizing rabbits with the purified GST-futions protein.Affinity-purified ALP antibody was obtained by binding serum IgG toALP-GST-fusion protein immobilized on glutathione-sepharose and elutingwith low pH and high salt.

EXAMPLE 5 Assay for ALP Activity Assay for Modulators of CatalyticActivity Materials and Methods

[0237] Recombinant wild-type and mutant ALP proteins are purified frombacteria as GST-fusion proteins. Lysates are bound to aglutathione-sepharose matrix and eluted with glutathione. The purifiedproteins are then washed with 2×1 mL HNTG, followed by one wash with 1mL of a buffer containing 100 mM 2-(N-morpholino)ethansulfonic acid(MES), pH 6.8, 150 mM NaCl, and 1 mM EDTA. The assay for phosphataseactivity is essentially done as described by Pei et al.(1993) usingp-nitrophenolphosphate (PNPP) as a generic PTP substrate. Briefly, afterthe last washing step, reactions are started by adding 50 mL AssayBuffer (100 mM MES pH 6.8, 150 mM NaCl, 10 mM DTT, 2 mM EDTA, and 50 mMp-nitrophenylphosphate) to the precipitates. Samples are incubated for20 min. at 23° C. The reactions are terminated by mixing 40 AL of eachsample (without beads) with 960 μL 1 N NaOH, and the absorbance ofp-nitrophenol was determined at 450 nm. To control for the presence ofALP in the precipitates, the precipitates are boiled in SDS samplebuffer and analyzed by SDS-PAGE. The presence of ALP is then detected byimmunoblot analysis with anti-ALP antibodies.

EXAMPLE 6 Screening Systems for the Identification of Inhibitors of ALPActivity

[0238] Assays may be performed in vitro or in vivo and are described indetail herein or can be obtained by modifying existing assays, such asthe growth assay described in patent application Ser. No. 08/487,088(Lyon & Lyon Docket No. 212/276), filed Jun. 7, 1995, by Tang et al.,and entitled “Novel Pharmaceutical Compounds,” or the assays describedin patent application Serial No. 60/005,167 (Lyon & Lyon Docket No.215/256), filed Oct. 13, 1995 by Seedorf et al., and entitled “Diagnosisand Treatment of TKA-1 related disorders,” all of which are herebyincorporated herein by reference in their entirety including anydrawings. Another assay which could be modified to use the genes of thepresent invention is described in International Application No. WO94/23039, published Oct. 13, 1994, hereby incorporated herein byreference in its entirety including any drawings. Other possibilitiesinclude detecting kinase activity in an autophosphorylation assay ortesting for kinase activity on standard substrates such as histones,myelin basic protein, gamma tubulin, or centrosomal proteins. Bindingpartners may be identified by putting the N-terminal portion of theprotein into a two-hybrid screen or detecting phosphotyrosine of a dualspecificity kinase (Fields and Song, U.S. Pat. No. 5,283,173, issuedFeb. 1, 1994, incorporated by reference herein, including any drawings).

[0239] One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. It will be readily apparentto one skilled in the art that varying substitutions and modificationsmay be made to the invention disclosed herein without departing from thescope and spirit of the invention.

[0240] All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which theinvention pertains. All patents and publications are herein incorporatedby reference to the same extent as if each individual publication wasspecifically and individually indicated to be incorporated by reference.

[0241] The invention illustratively described herein suitably may bepracticed in the absence of any element or elements, limitation orlimitations which is not specifically disclosed herein. Thus, forexample, in each instance herein any of the terms “comprising,”“consisting essentially of” and “consisting of” may be replaced witheither of the other two terms. The terms and expressions which have beenemployed are used as terms of description and not of limitation, andthere is no intention that in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the concepts herein disclosed may be resorted to bythose skilled in the art, and that such modifications and variations areconsidered to be within the scope of this invention as defined by theappended claims.

[0242] In addition, where features or aspects of the invention aredescribed in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup of members of the Markush group. Forexample, if X is described as selected from the group consisting ofbromine, chlorine, and iodine, claims for X being bromine and claims forX being bromine and chlorine are fully described.

[0243] In view of the degeneracy of the genetic code, other combinationsof nucleic acids also encode the claimed peptides and proteins of theinvention. For example, all four nucleic acid sequences GCT, GCC, GCA,and GCG encode the amino acide alanine. Therefore, if for an amino acidthere exists an average of three codons, a polypeptide of 100 aminoacids in length will, on average, be encoded by 3¹⁰⁰, or 5×10⁴⁷, nucleicacid sequences. Thus, a nucleic acid sequence can be modified to form asecond nucleic acid sequence, encoding the same polypeptide as endodedby the first nucleic acid sequences, using routine procedures andwithout undue experimentation. Thus, all possible nucleic acids thatencode the claimed peptides and proteins are also fully describedherein, as if all were written out in full taking into account the codonusage, especially that preferred in humans. Furthermore, changes in theamino acid sequences of polypeptides, or in the corresponding nucleicacid sequence encoding such polypeptide, may be designed or selected totake place in an area of the sequence where the significant activity ofthe polypeptide remains unchanged. For example, an amino acid change maytake place within a β-turn, away from the active site of thepolypeptide. Also changes such as deletions (e.g. removal of a segmentof the polypeptide, or in the corresponding nucleic acid sequenceencoding such polypeptide, which does not affect the active site) andadditions (e.g., addition of more amino acids to the polypeptidesequence without affecting the function of the active site, such as theformation of GST-fusion proteins, or additions in the correspondingnucleic acid sequence encoding such polypeptide without affecting thefunction of the active site) are also within the scope of the presentinvention. Such changes to the polypeptides can be performed by thosewith ordinary skill in the art using routine procedures and withoutundue experimentation. Thus, all possible nucleic and/or amino acidsequences that can readily be determined not to affect a significantactivity of the peptide or protein of the invention are also fullydescribed herein.

[0244] Other embodiments are within the following claims.

1 10 1 4456 DNA Artificial Sequence Description of Artificial SequenceRecombinant DNA sequence 1 ggcacgagag gagcagcaga agttcgggga gcgggttgcatacttccaga gcgccctgga 60 caagctcaat gaagccatca agttggccaa gggccagcctgacactgtgc aagacgcgct 120 tcgcttcact atggatgtca ttgggggaaa gtacaattctgccaagaagg acaacgactt 180 catttaccat gaggctgtcc cagcattgac acccttcagcctgtaaaagg agcccccttg 240 gtgaagccct tgccagtgaa ccccacagac ccagctgttacaggccctga catctttgcc 300 aaactggtac ccatggctgc ccacgaggcc tcgtcactgtacagtgagga gaaggccaag 360 ctgctccggg agatgatggc caagattgag gacaagaatgaggtcctgga ccagttcatg 420 gattcaatgc agttggatcc cgagacggtg gacaaccttgatgcctacag ccacatccca 480 ccccagctca tggagaagtg cgcggctctc agcgtccggcccgacactgt caggaacctt 540 gtacagtcca tgcaagtgct gtcaggtgtg ttcacggatgtggaggcttc cctgaaggac 600 atcagagatc tgttggagga ggatgagctg ctagagcagaagtttcagga ggcggtgggc 660 caggcagggg ccatctccat cacctccaag gctgagctggcagaggtgag gcgagaatgg 720 gccaagtaca tggaagtcca tgagaaggcc tccttcaccaacagtgagct gcaccgtgcc 780 atgaacctgc acgtcggcaa cctgcgcctg ctcagcgggccgcttgacca ggtccgggct 840 gccctgccca caccggccct ctccccagag gacaaggccgtgctgcaaaa cctaaagcgc 900 atcctggcta aggtgcagga gatgcgggac cagcgcgtgtccctggagca gcagctgcgt 960 gagcttatcc agaaagatga catcactgcc tcgctggtcaccacagacca ctcagagatg 1020 aagaagttgt tcgaggagca gctgaaaaag tatgaccagctgaaggtgta cctggagcag 1080 aacctggccg cccaggaccg tgtcctctgt gcactgacagaggccaacgt gcagtacgca 1140 gccgtgcggc gggtactcag cgacttggac caaaagtggaactccacgct gcagaccctg 1200 gtggcctcgt atgaagccta tgaggacctg atgaagaagtcgcaggaggg cagggacttc 1260 tacgcagatc tggagagcaa ggtggctgct ctgctggagcgcacgcagtc cacctgccag 1320 gcccgcgagg ctgcccgcca gcagctcctg gacagggagctgaagaagaa gccgccgcca 1380 cggcccacag ccccaaagcc gctgctgccc cgcagggaggagagtgaggc agtggaagca 1440 ggagaccccc ctgaggagct gcgcagcctc ccccctgacatggtggctgg cccacgactg 1500 cctgacacct tcctgggaag tgccaccccg ctccactttcctcccagccc cttccccagc 1560 tccacaggcc caggacccca ctatctctca ggccccttgccccctggtac ctactcgggc 1620 cccacccagc tgatacagcc cagggcccca gggccccatgcaatgcccgt agcacctggg 1680 cctgccctct acccagcccc tgcctacaca ccggagctgggccttgtgcc ccgatcctcc 1740 ccacagcatg gcgtggtgag cagtccctat gtgggggtagggccggcccc accagttgca 1800 ggtctcccct cggccccacc tcctcaattc tcaggccccgagttggccat ggcggttcgg 1860 ccagccacca ccacagtaga tagcatccag gcgcccatccccagccacac agccccacgg 1920 ccaaacccca cccctgctcc tcccccgccc tgcttccctgtgcccccacc gcagccactg 1980 cccacgcctt acacctaccc tgcaggggct aagcaacccatcccagcaca gcaccacttc 2040 tcttctggga tccccacagg ttttccagcc ccaaggattgggccccagcc ccagccccat 2100 cctcagcccc atccttcaca agcgtttggg cctcagcccccacagcagcc ccttccactc 2160 cagcatccac atctcttccc accccaggcc ccaggactcctacccccaca atccccctac 2220 ccctatgccc ctcagcctgg ggtcctgggg cagccgccaccccccctaca cacccagctc 2280 tacccaggtc ccgctcaaga ccctctgcca gcccactcaggggctctgcc tttccccagc 2340 cctgggcccc ctcagcctcc ccatccccca ctggcatatggtcctgcccc ttctaccaga 2400 cccatgggcc cccaggcagc ccctcttacc attcgagggccctcgtctgc tggccagtcc 2460 acccctagtc cccacctggt gccttcacct gccccatctccagggcctgg tccggtaccc 2520 cctcgccccc cagcagcaga accaccccct tgcctgcgccgaggcgccgc agctgcagac 2580 ctgctctcct ccagcccgga gagccagcat ggcggcactcagtctcctgg gggtgggcag 2640 cccctgctgc agcccaccaa ggtggatgca gctgagggtcgtcggccgca ggccctgcgg 2700 ctgattgagc gggaccccta tgagcatcct gagaggctgcggcagttgca gcaggagctg 2760 gaggcctttc ggggtcagct gggggatgtg ggagctctggacactgtctg gcgagagctg 2820 caagatgcgc aggaacatga tgcccgaggc cgttccatcgccattgcccg ctgctactca 2880 ctgaagaacc ggcaccagga tgtcatgccc tatgacagtaaccgtgtggt gctgcgctca 2940 ggcaaggatg actacatcaa tgccagctgc gtggaggggctctccccata ctgccccccg 3000 ctagtggcaa cccaggcccc actgcctggc acagctgctgacttctggct catggtccat 3060 gagcagaaag tgtcagtcat tgtcatgctg gtttctgaggctgagatgga gaagcaaaaa 3120 gtggcacgct acttccccac cgagaggggc cagcccatggtgcacggtgc cctgagcctg 3180 gcattgagca gcgtccgcag caccgaaacc catgtggagcgcgtgctgag cctgcagttc 3240 cgagaccaga gcctcaagcg ctctcttgtg cacctgcacttccccacttg gcctgagtta 3300 ggcctgcccg acagccccag caacttgctg cgcttcatccaggaggtgca cgcacattac 3360 ctgcatcagc ggccgctgca cacgcccatc attgtgcactgcagctctgg tgtgggccgc 3420 acgggagcct ttgcactgct ctatgcagct gtgcaggaggtggaggctgg gaacggaatc 3480 cctgagctgc ctcagctggt gcggcgcatg cggcagcagagaaagcacat gctgcaggag 3540 aagctgcacc tcaggttctg ctatgaggca gtggtgagacacgtggagca ggtcctgcag 3600 cgccatggtg tgcctcctcc atgcaaaccc ttggccagtgcaagcatcag ccagaagaac 3660 caccttcctc aggactccca ggacctggtc ctcggtggggatgtgcccat cagctccatc 3720 caggccacca ttgccaagct cagcattcgg cctcctggggggttggagtc cccggttgcc 3780 agcttgccag gccctgcaga gcccccaggc ctcccgccagccagcctccc agagtctacc 3840 ccaatcccat cttcctcccc accccccctt tcctccccactacctgaggc tccccagcct 3900 aaggaggagc cgccagtgcc tgaagccccc agctcggggcccccctcctc ctccctggaa 3960 ttgctggcct ccttgacccc agaggccttc tccctggacagctccctgcg gggcaaacag 4020 cggatgagca agcataactt tctgcaggcc cataacgggcaagggctgcg ggccacccgg 4080 ccctctgacg accccctcag ccttctggat ccactctggacactcaacaa gacctgaaca 4140 ggttttgcct acctggtcct tacactacat catcatcatctcatgcccac ctgcccacac 4200 ccagcagagc ttctcagtgg gcacagtctc ttactcccatttctgctgcc tttggccctg 4260 cctggcccag cctgcacccc tgtggggtgg aaatgtactgcaggctctgg gtcaggttct 4320 gctcctttat gggacccgac atttttcagc tctttgctattgaaataata aaccaccctg 4380 ttctgtgaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 4440 aaaaaaaaaa aaaaaa 4456 2 1274 PRT ArtificialSequence Description of Artificial Sequence ALP polypeptide sequence 2Met Ala Ala His Glu Ala Ser Ser Leu Tyr Ser Glu Glu Lys Ala Lys 1 5 1015 Leu Leu Arg Glu Met Met Ala Lys Ile Glu Asp Lys Asn Glu Val Leu 20 2530 Asp Gln Phe Met Asp Ser Met Gln Leu Asp Pro Glu Thr Val Asp Asn 35 4045 Leu Asp Ala Tyr Ser His Ile Pro Pro Gln Leu Met Glu Lys Cys Ala 50 5560 Ala Leu Ser Val Arg Pro Asp Thr Val Arg Asn Leu Val Gln Ser Met 65 7075 80 Gln Val Leu Ser Gly Val Phe Thr Asp Val Glu Ala Ser Leu Lys Asp 8590 95 Ile Arg Asp Leu Leu Glu Glu Asp Glu Leu Leu Glu Gln Lys Phe Gln100 105 110 Glu Ala Val Gly Gln Ala Gly Ala Ile Ser Ile Thr Ser Lys AlaGlu 115 120 125 Leu Ala Glu Val Arg Arg Glu Trp Ala Lys Tyr Met Glu ValHis Glu 130 135 140 Lys Ala Ser Phe Thr Asn Ser Glu Leu His Arg Ala MetAsn Leu His 145 150 155 160 Val Gly Asn Leu Arg Leu Leu Ser Gly Pro LeuAsp Gln Val Arg Ala 165 170 175 Ala Leu Pro Thr Pro Ala Leu Ser Pro GluAsp Lys Ala Val Leu Gln 180 185 190 Asn Leu Lys Arg Ile Leu Ala Lys ValGln Glu Met Arg Asp Gln Arg 195 200 205 Val Ser Leu Glu Gln Gln Leu ArgGlu Leu Ile Gln Lys Asp Asp Ile 210 215 220 Thr Ala Ser Leu Val Thr ThrAsp His Ser Glu Met Lys Lys Leu Phe 225 230 235 240 Glu Glu Gln Leu LysLys Tyr Asp Gln Leu Lys Val Tyr Leu Glu Gln 245 250 255 Asn Leu Ala AlaGln Asp Arg Val Leu Cys Ala Leu Thr Glu Ala Asn 260 265 270 Val Gln TyrAla Ala Val Arg Arg Val Leu Ser Asp Leu Asp Gln Lys 275 280 285 Trp AsnSer Thr Leu Gln Thr Leu Val Ala Ser Tyr Glu Ala Tyr Glu 290 295 300 AspLeu Met Lys Lys Ser Gln Glu Gly Arg Asp Phe Tyr Ala Asp Leu 305 310 315320 Glu Ser Lys Val Ala Ala Leu Leu Glu Arg Thr Gln Ser Thr Cys Gln 325330 335 Ala Arg Glu Ala Ala Arg Gln Gln Leu Leu Asp Arg Glu Leu Lys Lys340 345 350 Lys Pro Pro Pro Arg Pro Thr Ala Pro Lys Pro Leu Leu Pro ArgArg 355 360 365 Glu Glu Ser Glu Ala Val Glu Ala Gly Asp Pro Pro Glu GluLeu Arg 370 375 380 Ser Leu Pro Pro Asp Met Val Ala Gly Pro Arg Leu ProAsp Thr Phe 385 390 395 400 Leu Gly Ser Ala Thr Pro Leu His Phe Pro ProSer Pro Phe Pro Ser 405 410 415 Ser Thr Gly Pro Gly Pro His Tyr Leu SerGly Pro Leu Pro Pro Gly 420 425 430 Thr Tyr Ser Gly Pro Thr Gln Leu IleGln Pro Arg Ala Pro Gly Pro 435 440 445 His Ala Met Pro Val Ala Pro GlyPro Ala Leu Tyr Pro Ala Pro Ala 450 455 460 Tyr Thr Pro Glu Leu Gly LeuVal Pro Arg Ser Ser Pro Gln His Gly 465 470 475 480 Val Val Ser Ser ProTyr Val Gly Val Gly Pro Ala Pro Pro Val Ala 485 490 495 Gly Leu Pro SerAla Pro Pro Pro Gln Phe Ser Gly Pro Glu Leu Ala 500 505 510 Met Ala ValArg Pro Ala Thr Thr Thr Val Asp Ser Ile Gln Ala Pro 515 520 525 Ile ProSer His Thr Ala Pro Arg Pro Asn Pro Thr Pro Ala Pro Pro 530 535 540 ProPro Cys Phe Pro Val Pro Pro Pro Gln Pro Leu Pro Thr Pro Tyr 545 550 555560 Thr Tyr Pro Ala Gly Ala Lys Gln Pro Ile Pro Ala Gln His His Phe 565570 575 Ser Ser Gly Ile Pro Thr Gly Phe Pro Ala Pro Arg Ile Gly Pro Gln580 585 590 Pro Gln Pro His Pro Gln Pro His Pro Ser Gln Ala Phe Gly ProGln 595 600 605 Pro Pro Gln Gln Pro Leu Pro Leu Gln His Pro His Leu PhePro Pro 610 615 620 Gln Ala Pro Gly Leu Leu Pro Pro Gln Ser Pro Tyr ProTyr Ala Pro 625 630 635 640 Gln Pro Gly Val Leu Gly Gln Pro Pro Pro ProLeu His Thr Gln Leu 645 650 655 Tyr Pro Gly Pro Ala Gln Asp Pro Leu ProAla His Ser Gly Ala Leu 660 665 670 Pro Phe Pro Ser Pro Gly Pro Pro GlnPro Pro His Pro Pro Leu Ala 675 680 685 Tyr Gly Pro Ala Pro Ser Thr ArgPro Met Gly Pro Gln Ala Ala Pro 690 695 700 Leu Thr Ile Arg Gly Pro SerSer Ala Gly Gln Ser Thr Pro Ser Pro 705 710 715 720 His Leu Val Pro SerPro Ala Pro Ser Pro Gly Pro Gly Pro Val Pro 725 730 735 Pro Arg Pro ProAla Ala Glu Pro Pro Pro Cys Leu Arg Arg Gly Ala 740 745 750 Ala Ala AlaAsp Leu Leu Ser Ser Ser Pro Glu Ser Gln His Gly Gly 755 760 765 Thr GlnSer Pro Gly Gly Gly Gln Pro Leu Leu Gln Pro Thr Lys Val 770 775 780 AspAla Ala Glu Gly Arg Arg Pro Gln Ala Leu Arg Leu Ile Glu Arg 785 790 795800 Asp Pro Tyr Glu His Pro Glu Arg Leu Arg Gln Leu Gln Gln Glu Leu 805810 815 Glu Ala Phe Arg Gly Gln Leu Gly Asp Val Gly Ala Leu Asp Thr Val820 825 830 Trp Arg Glu Leu Gln Asp Ala Gln Glu His Asp Ala Arg Gly ArgSer 835 840 845 Ile Ala Ile Ala Arg Cys Tyr Ser Leu Lys Asn Arg His GlnAsp Val 850 855 860 Met Pro Tyr Asp Ser Asn Arg Val Val Leu Arg Ser GlyLys Asp Asp 865 870 875 880 Tyr Ile Asn Ala Ser Cys Val Glu Gly Leu SerPro Tyr Cys Pro Pro 885 890 895 Leu Val Ala Thr Gln Ala Pro Leu Pro GlyThr Ala Ala Asp Phe Trp 900 905 910 Leu Met Val His Glu Gln Lys Val SerVal Ile Val Met Leu Val Ser 915 920 925 Glu Ala Glu Met Glu Lys Gln LysVal Ala Arg Tyr Phe Pro Thr Glu 930 935 940 Arg Gly Gln Pro Met Val HisGly Ala Leu Ser Leu Ala Leu Ser Ser 945 950 955 960 Val Arg Ser Thr GluThr His Val Glu Arg Val Leu Ser Leu Gln Phe 965 970 975 Arg Asp Gln SerLeu Lys Arg Ser Leu Val His Leu His Phe Pro Thr 980 985 990 Trp Pro GluLeu Gly Leu Pro Asp Ser Pro Ser Asn Leu Leu Arg Phe 995 1000 1005 IleGln Glu Val His Ala His Tyr Leu His Gln Arg Pro Leu His Thr 1010 10151020 Pro Ile Ile Val His Cys Ser Ser Gly Val Gly Arg Thr Gly Ala Phe1025 1030 1035 1040 Ala Leu Leu Tyr Ala Ala Val Gln Glu Val Glu Ala GlyAsn Gly Ile 1045 1050 1055 Pro Glu Leu Pro Gln Leu Val Arg Arg Met ArgGln Gln Arg Lys His 1060 1065 1070 Met Leu Gln Glu Lys Leu His Leu ArgPhe Cys Tyr Glu Ala Val Val 1075 1080 1085 Arg His Val Glu Gln Val LeuGln Arg His Gly Val Pro Pro Pro Cys 1090 1095 1100 Lys Pro Leu Ala SerAla Ser Ile Ser Gln Lys Asn His Leu Pro Gln 1105 1110 1115 1120 Asp SerGln Asp Leu Val Leu Gly Gly Asp Val Pro Ile Ser Ser Ile 1125 1130 1135Gln Ala Thr Ile Ala Lys Leu Ser Ile Arg Pro Pro Gly Gly Leu Glu 11401145 1150 Ser Pro Val Ala Ser Leu Pro Gly Pro Ala Glu Pro Pro Gly LeuPro 1155 1160 1165 Pro Ala Ser Leu Pro Glu Ser Thr Pro Ile Pro Ser SerSer Pro Pro 1170 1175 1180 Pro Leu Ser Ser Pro Leu Pro Glu Ala Pro GlnPro Lys Glu Glu Pro 1185 1190 1195 1200 Pro Val Pro Glu Ala Pro Ser SerGly Pro Pro Ser Ser Ser Leu Glu 1205 1210 1215 Leu Leu Ala Ser Leu ThrPro Glu Ala Phe Ser Leu Asp Ser Ser Leu 1220 1225 1230 Arg Gly Lys GlnArg Met Ser Lys His Asn Phe Leu Gln Ala His Asn 1235 1240 1245 Gly GlnGly Leu Arg Ala Thr Arg Pro Ser Asp Asp Pro Leu Ser Leu 1250 1255 1260Leu Asp Pro Leu Trp Thr Leu Asn Lys Thr 1265 1270 3 23 DNA ArtificialSequence Description of Artificial Sequence Primer 3 gayttytggvrnatgrtntg gga 23 4 23 DNA Artificial Sequence Description of ArtificialSequence Primer 4 cggccsaync cngcnswrca rtg 23 5 26 DNA ArtificialSequence Description of Artificial Sequence Primer 5 atccccggctctgaytayat hmaygc 26 6 8 PRT Artificial Sequence Description ofArtificial Sequence Synthetic peptide 6 Asp Phe Trp Xaa Met Xaa Trp Xaa1 5 7 7 PRT Artificial Sequence Description of Artificial SequenceSynthetic peptide 7 His Cys Xaa Ala Gly Xaa Gly 1 5 8 9 PRT ArtificialSequence Description of Artificial Sequence Synthetic peptide 8 Ile ProGly Ser Asp Tyr Ile Xaa Ala 1 5 9 5 PRT Unknown Organism Description ofUnknown Organism Illustrative motif 9 His Cys Ser Ala Gly 1 5 10 5 PRTUnknown Organism Description of Unknown Organism Illustrative motif 10His Cys Ser Ser Gly 1 5

What is claimed is:
 1. An isolated, enriched or purified nucleic acidmolecule encoding an ALP polypeptide.
 2. The nucleic acid molecule ofclaim 1, wherein said nucleic acid molecule comprises a nucleotidesequence that (a) encodes a polypeptide having the full length aminoacid sequence set forth in SEQ ID NO:2; (b) is the complement of thenucleotide sequence of (a); (c) hybridizes under highly stringentconditions to the nucleotide molecule of (a) and encodes a naturallyoccurring ALP polypeptide; (d) encodes an ALP polypeptide having thefull length amino acid sequence of the sequence set forth in SEQ IDNO:2, except that it lacks one or more of the following segments ofamino acid residues: 1-857, 353-777, 858-1096, 1097-1274, 1101-1214 ofSEQ ID NO:2; (e) is the complement of the nucleotide sequence of (d);(f) encodes a polypeptide having the amino acid sequence set forth inSEQ ID NO:2 from amino acid residues 1-857, 353-777, 858-1096,1097-1274, 1101-1214 of SEQ ID NO:2; (g) is the complement of thenucleotide sequence of (f); (h) encodes a polypeptide having the fulllength amino acid sequence set forth in SEQ ID NO:2, except that itlacks one or more of the domains selected from the group consisting of aan N-terminal domain, an N-terminal proline-rich domain, a catalyticdomain, a C-terminal proline/serine-rich domain, and a C-terminaldomain; or (i) is the complement of the nucleotide sequence of (h). 3.The nucleic acid molecule of claim 1, wherein said nucleic acid moleculeis isolated, enriched, or purified from a mammal.
 4. The nucleic acidmolecule of claim 3, wherein said mammal is a human.
 5. The nucleic acidmolecule of claim 1, further comprising a vector or promoter effectiveto initiate transcription in a host cell.
 6. A nucleic acid probe forthe detection of nucleic acid encoding an ALP polypeptide in a sample.7. The probe of claim 6 wherein said polypeptide comprises at least 6contiguous amino acids of the amino acid sequence shown in SEQ ID NO:2.8. A nucleic acid molecule comprising one or more regions that encode anALP polypeptide or an ALP domain polypeptide, wherein said ALPpolypeptide or said ALP domain polypeptide is fused to a non-ALPpolypeptide.
 9. A recombinant cell comprising a nucleic acid moleculeencoding either (a) an ALP polypeptide; (b) an ALP domain polypeptide;or (c) an ALP polypeptide or ALP domain polypeptide fused to a non-ALPpolypeptide.
 10. An isolated, enriched or purified ALP polypeptide. 11.The polypeptide of claim 10, wherein said polypeptide is a fragment ofthe protein encoded by the full length amino acid sequence set forth inSEQ ID NO:2.
 12. The polypeptide of claim 10, wherein said polypeptidecomprises an amino acid sequence having (a) the full length amino acidsequence set forth in SEQ ID NO:2; (b) the full length amino acidsequence of the sequence set forth in SEQ ID NO:2, except that it lacksone or more of the following segments of amino acid residues: 1-857,353-777, 858-1096, 1097-1274, 1101-1214 of SEQ ID NO:2; (c) the aminoacid sequence set forth in SEQ ID NO:2 from amino acid residues 1-857,353-777, 858-1096, 1097-1274, 1101-1214 of SEQ ID NO:2; or (d) the fulllength amino acid sequence set forth in SEQ ID NO:2 except that it lacksone or more of the domains selected from the group consisting of anN-terminal domain, an N-terminal proline-rich domain, a catalyticdomain, a C-terminal proline/serine-rich domain, and a C-terminaldomain.
 13. An antibody or antibody fragment having specific bindingaffinity to an ALP polypeptide or an ALP domain polypeptide.
 14. Ahybridoma which produces an antibody having specific binding affinity toan ALP polypeptide.
 15. A method for identifying a substance capable ofmodulating ALP activity comprising the steps of: (a) contacting an ALPpolypeptide with a test substance, and (b) determining whether saidsubstance alters the activity of said polypeptide.
 16. A method foridentifying a substance capable of modulating ALP activity in a cellcomprising the steps of: (a) expressing an ALP polypeptide in a cell,(b) adding a test substance to said cells, and (c) monitoring a changein cell phenotype, cell proliferation, cell differentiation, ALPcatalytic activity, or the interaction between an ALP polypeptide and anatural binding partner.
 17. A method of preventing or treating anabnormal condition by administering to a patient in need of suchtreatment a compound that modulates the function of an ALP polypeptide.18. The method of claim 17, wherein said abnormal condition involves anabnormality in ALP signal transduction pathway.
 19. The method of claim18, wherein said abnormal condition is cancer.
 20. The method of claim17, wherein said compound modulates the function of an ALP polypeptidein vitro.
 21. A kit, comprising the compound of claim 17 and a protocolfor the use of said compound.
 22. The kit of claim 21, wherein saidprotocol is approved by the Food and Drug Administration.